1. PCR ARRAY
.
GUIDE v4 0
The Complete Technical Reference to PCR ARRAYS
Cancer
Cytokines
Biomarkers
ECM & Adhesion
Oxidative Stress
Signal Transduction
Inflammation
Stem Cells
MicroRNA
Epigenetics
Toxicology
NEW 100 Selected Peer-reviewed Publications
A QIAGEN Company
Focus on your PathwayTM
2.
3. Table of Contents
PCR ARRAY Guide v4.0
PCR ARRAY GUIDE version 4.0
RT2 ProfilerTM PCR ARRAYS ................................................................................................................2
The Most Accurate and Sensitive Technology for Pathway-Powered Gene Expression Analysis
RT2 Profiler PCR ARRAY System .....................................................................................................4
A Complete and Reliable Solution for Gene Expression Analysis
Custom PCR ARRAYS & Assay Design ................................................................................................5
Focus on Your Genes with PCR Arrays Created from Your Gene List
RT2 SYBR Green qPCR Master Mixes ..............................................................................................6
®
The Required High-Performance Master Mix for RT2 Profiler PCR Arrays
RT2 Profiler PCR ARRAY Performance ..........................................................................................7
Reliable and Reproducible Pathway-focused Gene Expression Analysis
RT2 Nano PreAMPTM cDNA Synthesis Technology ..........................................................................8
Enabling the Analysis of One Nanogram of RNA with RT2 Profiler PCR Arrays
RT2 FFPE PreAMP cDNA Synthesis Technology............................................................................9
Enabling the Analysis of FFPE Samples with RT2 Profiler PCR Arrays
PCR ARRAY Data Analysis Guide ..................................................................................................10
Complete Solutions for Interpreting RT2 Profiler PCR Array Data
Service Core for PCR ARRAY Gene Expression Analysis ..................................................10
Complete Set of Quick and Convenient Gene Expression Profilng Services
QIAGEN RNeasy Kits ........................................................................................................................11
®
Purification of High-quality Total RNA from a Range of Biological Samples
QIAGEN Products for Sample Disruption ................................................................................12
Fast and Effective Disruption of Biological Samples at a Range of Throughputs
QIAGEN Products for RNA Stabilization ................................................................................13
Convenient and Immediate Stabilization of RNA in a Range of Biological Samples
Research Area-Focused PCR ARRAY Product Listing ...........................................................14
Search Our Complete Catalog of PCR Array Products by Your Area of Research
RT2 MicroRNA PCR ARRAYS ................................................................................................................16
Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA
QIAGEN miRNeasy Kits ....................................................................................................................18
Purification of Total RNA from a Range of Biological Samples
ChampionChIPTM PCR System .............................................................................................................19
Reliable Chromatin IP with Real-time PCR Precision Achieved in Only One Day
Methyl-ProfilerTM PCR ARRAY System ..........................................................................................20
Simple, Fast and Reliable DNA Methylation Analysis Without Bisulfite Conversion
QIAGEN DNeasy Blood and Tissue Kits ...................................................................................22
®
Purification of Total DNA from Cells, Tissues and Blood
QIAGEN QIAamp DNA Mini Kits ....................................................................................................22
®
Purification of Genomic, Mitochondrial, Bacterial, Parasitic, or Viral DNA
PreAnalytiX PAXgene DNA System .............................................................................................23
®
®
Collection & Stabilization of Blood or Tissue Samples & Subsequent DNA Purification
PCR ARRAY Published Literature ...............................................................................................24
Publications Citing RT2 Profiler PCR Arrays
Complete Product Catalog Index .................................................................................................27
Search the Complete Catalog of RT2 Profiler PCR Array Products and Accessories
www.SABiosciences.com
1
4. RT2 Profiler PCR ARRAYS
TM
The Most Accurate and Sensitive Technology for Pathway Gene Expression Analysis
What Are PCR ARRAYS?
How PCR ARRAYS Work
RT² ProfilerTM PCR Arrays are the most reliable and sensitive gene expression
profiling technology for analyzing focused panels of genes in signal transduction,
biological process, or disease-related pathways using real-time PCR.
Each cataloged PCR Array contains a list of the pathway-focused genes as well
as five housekeeping (refererence) genes on the array. Wells H6 through H12
contain a panel of proprietary controls to monitor genomic DNA contamination
(HGDC) as well as the first strand synthesis (RTC) and real-time PCR efficiency
(PPC).
2. Convert Total RNA to cDNA.
Control
Experimental
3. Add cDNA to RT2 SYBR® Green Master Mix.
Aliquot Mixture Across PCR Array.
Why Use RT Profiler PCR ARRAYS?
2
1. Isolate RNA from Cells, Tissues, FFPE and/or Blood.
TM
Simplicity:
The simplicity of RT2 Profiler PCR Arrays makes routine expression profiling
practical in any research laboratory with a real-time instrument.
4. Run in Your Real-Time PCR Instrument.
Performance:
Experimental Profile
Control Profile
1.E-000
Delta Rn
Profiler PCR Arrays have the sensitivity, reproducibility, specificity, and
reliability to accurately profile multiple genes simultaneously in 96- or
384-well formats.
1.E+001
1.E-000
Delta Rn
1.E+001
RT²
1.E-001
1.E-003
1.E-003
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Relevance:
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle Number
Cycle Number
5. Data Analysis.
10-6
p-Value for Fold Change
Anatomy of a 96-well RT Profiler PCR ARRAY
2
1.E+00
A
B
A
1.E-01
10-5
Normal Breast
RT² Profiler PCR Arrays focus on profiling the genes relevant to the pathways
or disease states important to your research.
10-4
10-3
10-2
0
1
2
3
4
CCL2
CCL20 CCL21 CCL23 CCL24 CCL25 CCL26
CCL3
CCL4
CCL8
CCR1
CCR2
CCR8
CCR9 CEBPB
CCR3
C5
CCL1
Breast Tumor
CCR4
CCR5
CCL11 CCL13 CCL15 CCL16 CCL17 CCL18
CCR6
CCR7
CCL5
CCL7
IL10RA IL10RB
IL13 IL13RA1 IL17C
IL1A
IL1F6
IL1F7
IL1F8
IL1F9
IL1R1
IL1RN
IL22
IL5
IL5RA
IL9
IL9R
LTA
LTB
LTB4R
MIF
SCYE1
SPP1
TNF
IL1F10
IL1B
IL8RA
IL8
16
CRP
CX3CR1 CXCL1 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL2 CXCL3 CXCL5 CXCL6 CXCL9
IL10
SYBR® Green Versus TaqMan® Chemistries
IL1F5
IL8RB
TaqMan Log2 FC
C4A
1.E+00
-1
TIMP3
1.E-01
CCL19
C3
MCAM
ITGB4
1.E-02
-2
MMP9
ITGA2
1.E-03
-3
1.E-06
D
2
BCL6
TGFB1
ITGB3
1.E-04
1.E-04
C
Fold Change Ratio (log )
ABCF1
1.E-03
1.E-05
-4
CDKN2A
FGFR2
1.E-06
1
CCNE1
1.E-02
1.E-05
10-1
D
ICEBERG IFNA2
1.E-001
1.E-002
1.E-002
Figure 2: Comparable Biological Results.* Gene expression
12
E = 0.97
R2 = 0.99
8
4
-16
-12
-8
-4
4
-4
8
12
16
-8
86 GENES
-12
-16
TNFSF5 TOLLIP XCR1
analysis was compared between RT2
Profiler PCR Arrays (SYBR Green-based)
and the TaqMan platform. Regression
analysis of fold differences, with data
normalized against POLR2A, demonstrate that both platforms yield similar
biological results.
RT2 PCR Log2 FC
HPRT1 RPL13A GAPDH ACTB
HOUSEKEEPING
(REFERENCE) GENES
HGDC
RTC
RTC
RTC
GENOMIC REVERSE TRANSCRIPTION
CONTROLS
DNA CONTROL
PPC
PPC
PPC
25
POSITIVE
PCR CONTROLS
Figure 1: Each Well in a PCR Array Measures the Expression of a Gene Related to a
Pathway or Disease State. The Human Inflammatory Cytokines and Receptors PCR Array in a
96-well format is shown. This is also available in a 384-well format.
Figure 3: Sensitivity with RT2
SYBR Green Versus TaqMan
Chemistry.* PCR amplicons detected
TaqMan
20
Ct
B2M
E = 100%
R2 = 0.9998
using the same primer pair with or
without TaqMan probes in either SYBR
Green or TaqMan chemistry. SYBR green
chemistry yields earlier Cts for each
dilution, demonstrating better sensitivity
than TaqMan chemistry.
15
SYBR Green
10
E = 100%
R2 = 0.9999
5
4
2
Support@SABiosciences.com
6
8
10
Log [Copy Number]
12
* BMC Genomics 2008, 9: 378.
5. RT2 ProfilerTM PCR ARRAYS
PCR ARRAY Guide v4.0
Application: Angiogenesis
Application: ECM PCR ARRAYS for Cancer Biomarker Discovery
ECM and Cell Adhesion
VEGF Regulation of Cellular Architecture
0
100
-50
10-1
Breast Tumor
101
-100
10-2
-150
-200
10-3
UP
-R
EG
UL
AT
ED
10-5
Chen, C. et al. Cancer Research. 2009; 69 (16): 6721-6729.
COL4A2
CTNND2
ADAMTS1
SELE
TIMP3
NO
10-5
ITGB4
MMP9
MMP3
10-4
ANGPT1
FGF1
KDR
TIMP3
FIGF
SERPINF1
JAG1
FGFR3
B2M
TNFA
ECGF1
CXCL10
LAMA5
TIMP1
TGFA
IL1B
VEGFA
ACTB
TGFB1
SPHK1
VEGFC
FGF2
MDK
PTGS1
ENG
IL6
PECAM1
THBS1
EREG
NRP2
MMP9
PGF
IL8
Fold Change
mAb225/lgG
50
FN1
DO
WN
-R
EG
UL
AT
ED
CNTN1
ITGB3
GE
AN
CH
10-3
10-4
10-2
10-1
101
100
Normal Breast
Figure 4: Relative Fold Change Between Disorganized and Organized Colonies
Using the RT2 Profiler Angiogenesis PCR Array. RNA isolated from unorganized T4-2 cells
treated with a control antibody (IgG) or reverted to an organized colony by blocking EGFR signaling
(mAB225) was reverse transcribed and relative gene expression data was obtained using the Human
Angiogenesis PCR Arrays. The expression profile of 84 genes relevant to Angiogenesis as well as 5
housekeeping genes was assayed. Fold change calculations were done using SABiosciences’ data
analysis software which automatically calculates the fold change in gene expression between the
treated and control groups.
Figure 7: ECM and Cell Adhesion PCR Arrays Revealed Up- and Down-Regulated
Genes in Breast Cancer. Total RNA from a normal human breast and a human breast tumor were
characterized in technical triplicates, and the relative expression levels for each gene in the two samples
are plotted against each other in the Scatter Plot. Genes encoding the matrix metallopeptidases (MMP3
and MMP9) and their inhibitors (TIMP3) are up-regulated, while genes encoding integrins (ITGB3 and
ITGB4) are down-regulated, by at least three-fold (outside the silver field) in breast tumors relative to
normal tissue.
p value
STATISTICAL SIGNIFICANCE
Application: Immune Response
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
100
Popular Pathway-Focused PCR ARRAYS
Common Cytokines
IL10
IL1A
IL1B
CSF1
PDGFA
TGFB2
TNFSF11
BMP6
TNFRSPSF11B
TNFSF14
TNFSF13B
IFNA5
IFNG CSF2 IL9
IL11
IL21
IL5
IL13
TNF
LTA
TNFSF10
IL1F7
IL2
IL22
IL3
IL17
BMP3
-7
-5
-3
-1
1
3
5
7
9
fold difference [log2]
DOWN-REGULATED
11
13
15
17
UP-REGULATED
Figure 5: Common Cytokine PCR Array Identified 23 Up-Regulated and 6 DownRegulated Genes Following PBMC Stimulation. Triplicate total RNA samples from human
peripheral blood mononuclear cells (either untreated or stimulated with 50 ng/mL PMA and 1 µg/mL
ionomycin for 6 hours) were characterized with the Human Common Cytokine PCR Array. Twenty-three
cytokine genes are up-regulated (> 5-fold,p < 0.0005) including interleukins, colony stimulating factors,
and TNF ligands after 6 hours of stimulation. Six interleukin and TNF ligand genes are down-regulated
under the same conditions.
Application: Determining Drug Toxicity with PCR ARRAYS
Stress and Toxicity PathwayFinder
5005
128
Inflammatory Cytokines & Receptors
NFκB Signaling Pathway
Oxidative Stress & Antioxidant Defense
Signal Transduction PathwayFinderTM
Stem Cell (Embryonic, Mesenchymal)
Stress and Toxicity PathwayFinderTM
TGFβ / BMP Signaling Pathway
Th1-Th2-Th3
Toll-Like Receptor Signaling Pathway
Wnt Signaling Pathway
Complete PCR Array List
Custom PCR Arrays
www.SABiosciences.com/ArrayList.php
(Detailed Information on Page 5)
Complete RT Profiler PCR ARRAY System
2
Product
Plate Format
RT Profiler PCR Array
2
96-well Plate
384-well Plate
Product
RT First Strand cDNA Synthesis Kit
2
RT SYBR Green w/ ROX Master Mix
2
RT SYBR Green w/ Fluorescein Master Mix
2
RT SYBR Green Only Master Mix (see page 6)
HSPA6
CRYAB
CSF2
HSPA1A
TNF
DDIT3
HSPH1
CYP1A1
HSPA5
HSPCA
2
DNAJB4
HMOx1
30
20
10
0
119
1GADD45A
125
1ACTB
ActosTM
Avandia®
Rezulin®
MT2A
5000
18SrRNA
Fold Up-Regulation
tm
Angiogenesis
Apoptosis
Cancer PathwayFinderTM
Cell Cycle
Chemokines and Receptors
Common Cytokines
DNA Damage Signaling Pathway
Endothelial Cell Biology
Epithelial to Mesenchymal Transition
Extracellular Matrix and Adhesion
Figure 6: Stress and Toxicity PathwayFinderTM PCR Array Uncovered Distinct Gene
Expression Profiles Associated with Liver Toxicity Caused by 3 PPARγ Agonists.
RNA from HepG2 cells treated with three different glitazone PPARγ agonists for type 2 diabetes mellitus
was characterized, and the results were compared to that of a vehicle (DMSO) control. The drug
withdrawn due to idiosyncratic liver toxicity (Rezulin), induces very different changes in the expression
of stress-related genes than two safer drugs still on the market (Avandia and Actos).
Pack Size
2, 12, 24 Arrays
4 Arrays
Catalog #
Pack Size
12 Samples
330401
2, 12, 24 Arrays
2, 12, 24 Arrays
2, 12, 24 Arrays
Various
Various
Various
PCR Array Data Analysis Software
FREE
PCR ARRAY Accessories
Pack Size
Catalog #
RT Nano PreAMP cDNA Synthesis Kit
2
RT FFPE PreAMP cDNA Synthesis Kit
2
RT Nano PreAMP cDNA Synthesis Primer Mixes
12 Samples
12 Samples
12 Samples
330451
330461
Inquire
2
www.SABiosciences.com
3
6. RT2 Profiler PCR ARRAY System
A Complete & Reliable Solution for Gene Expression Analysis
How The PCR ARRAY System Works
Cells, Tissues,
FFPE, Blood,
and Biofluids
RT 2 Profiler PCR ARRAYS
Each pathway-focused PCR Array includes 89-wet bench validated qPCR Primers
Assays (including 5 housekeeping genes) and a proprietary control panel.
RNeasy
KITS
RT 2 SYBR Green qPCR Master Mixes
A unique formulation of buffers that co-evolved with the primer design algorithm
provides high amplification efficiencies. Available with reference dyes (ROX,
Fluorescein or without).
Total RNA
gDNA
Elimination
Solution (GE)
RT Enzyme
Oligo-dTs
Random
Hexamers
External
RNA
Control
(RTC)
dNTPs
RT2 FIRST STRAND cDNA SYNTHESIS KIT
GENOMIC DNA
ELIMINATION
REVERSE TRANSCRIPTION
RT 2 First Strand cDNA Synthesis Kit
RE SOLUTION
5 MINUTES
15 MINUTES
AAAAA
AAAAA
AAAAA
AAAAA
First Strand cDNA
An External RNA Control detected by the PCR Array tests the quality of input RNA.
It also features a proprietary genomic DNA elimination buffer essential for
eliminating residual gDNA, ensuring specific detection of mRNA.
FREE Data Analysis Software
The power of the PCR Array to assess the expression of a pathwayfocused set of genes over a wide range of detection yields an abundance
of data. With our FREE PCR Array Data Analysis tool, go from raw Ct
values to fold change results displayed in a variety of formats (Scatter
Plots, Volcano Plots, Clustergram) in a MATTER OF MINUTES.
45
GE Effectively Removes Genomic DNA
40
CLEAN OF gDNA
35
Ct GDC
RNA
ISOLATION
5-120 MINS
SABiosciences RT2 Profiler PCR Arrays are a complete system for PathwayFocused Gene Expression Analysis. From Sample Preparation to Data Analysis,
the PCR Array system includes four components that GUARANTEE high-quality,
reproducible, and reliable gene expression data.
Integral to the performance of the PCR Array system is a proprietary set of control
elements that enhance the reliability of your data and serve as a guarantee for
performance over time. These elements allow researchers to quickly assess the
quality of their data by determining if samples were contaminated with genomic
DNA (gDNA), the quality of the reverse transcription reaction, and real-time PCR
efficiency. Each component of the RT2 Profiler system contributes to these quality
control elements by incorporating an interlocked system for comprehensive
monitoring of each step of the PCR Array process.
Untreated
GE Treated
30
25
20
dNTPs
SYBR
Green
Dye
DNA
Polymerase
Binding
Elongation
SYBR Green
Binding
Detection
HEK293T Cells
Mouse Spinal Tissue
Mouse Brain Tissue
RNA Source
Rat Brain tissue
Figure 1: Elimination of Genomic DNA Contamination. RNA from HEK 293T cells, mouse
spinal tissue, mouse brain tissue, or rat brain tissue was characterized on SYBR Green PCR Arrays before
(blue bars) and after (red bars) treatment with gDNA Elimination Buffer from the RT² First Strand Kit.
Average Ct (RTC-PPC)
Hot-Start DNA
Polymerase
RT2 SYBR GREEN
MASTER MIX
10
HEAT
ACTIVATION
CYCLING AND DETECTION
2 HOURS (40 CYCLES)
10 MINS
15
10
Reverse Transcription Monitoring
8
6
FAIL
PASS
4
2
0
None
Mg2+
Addition / Treatment
TRIzol®
Figure 2: Monitoring Inhibition in Reverse Transcription. Human universal RNA was
added with magnesium salt to simulate RNA degradation or added with TRIzol® reagent to simulate
contamination that inhibits enzyme activity. RT² First Strand Kit was used for cDNA synthesis.
4
Support@SABiosciences.com
7.
8. RT2 SYBR® Green qPCR Master Mixes
The Required High-Performance Master Mix for RT2 Profiler PCR Arrays
SYBR Green Detection is a popular approach used in quantifying gene expression
analysis with RT-PCR. It relies on the preferential binding of the SYBR green dye
to double-stranded DNA, resulting in strong fluorescence emission signals, with
the signal intensity proportional to the amount of double-stranded DNA present.
Greater Sensitivity Without Sacrificing Specificity
2
RT SYBR Green qPCR Master Mix
Primer
SYBR Green
DNA Template
MMP15
Gel
MMP13
Gel
Competitor I Master Mix
Polymerase
High quality PCR reaction components are essential for achieving superior
amplification specificity and efficiency. SABiosciences offers a complete solution for using SYBR Green PCR Arrays with the RT2 SYBR Green qPCR Master
Mixes. Each mix includes a Hot Start Taq DNA polymerase, which provides
tighter control over activity, and other proprietary chemical components that
significantly minimize primer dimer formation, thereby enhancing amplification
efficiencies for even the most difficult-to-amplify genes. The higher SYBR Green
signal from our formulations provides greater sensitivity and ensures clean
results without sacrificing specificity or amplification efficiency.
Brighter SYBR Green Signal
MMP15
Gel
MMP13
Gel
Figure 2: RT2 SYBR Green qPCR Master Mixes Provide Greater Sensitivity Without
Sacrificing Specificity. RT2 SYBR Green qPCR Master Mixes and Competitor I Master Mixes were
2
used in qPCR assays to detect the human MMP13 and MMP15 mRNA in reference RNA. RT SYBR Green
qPCR Master mixes provide detection of the genes at an earlier threshold cycle value (Ct). The real-time
dissociation curves and agarose gel electrophoresis characterization reveal the presence of a non-specific
2
secondary product generated with the competitor’s master mix which is not amplified by the RT SYBR
Green qPCR Master Mix.
2
RT SYBR Green qPCR Master Mix from SABiosciences
A
Amplification Plots
10
Compatible PCR Instruments
Applied Biosystems (ABI): 5700, 7000, 7300, 7500, 7500 FAST, 7700, 7900HT,
StepOnePlus (96- and 384-well blocks)
Bio-Rad: CFX96, CFX384, iCycler, iQ5, MyiQ, MyiQ 2, Chromo4, Opticon 2
Stratagene: Mx3000P, Mx3005P, Mx4000
Roche: LightCycler 480 (96- and 384-well blocks)
Eppendorf: Mastercycler ep realplex 2/2S, 4/4S
TaKaRa: TP-800
∆RN
8
6
4
2
0
Signal [-d(RFU)/dT]
B
4
0
8
12
16
20
24
Cycle Number
28
32
36
40
RT qPCR Master Mixes
2
Dissociation Curves
0.5
Product
RT SYBR Green w/ ROX
qPCR Master Mix
2
0.4
0.3
0.2
RT SYBR Green w/ Fluorescein
qPCR Master Mix
2
0.1
0
60
65
70
75
80
85
Melting Temperature [oC]
90
95
RT SYBR Green qPCR Master Mix
2
Figure 1: RT2 SYBR Green qPCR Master Mixes Provide Greater Sensitivity with a
Brighter SYBR Green Signal. Four commercial master mixes were used to detect the expression
of human ACTB from the same universal reference RNA. The amplification (A) and the dissociation
curves (B) for the master mix from SABiosciences demonstrate a sharper amplification curve and a
brighter SYBR Green signal than observed with three competing master mixes.
6
Support@SABiosciences.com
Without Reference Dye
* Plate format is 384-well.
Size
Catalog #
SABio #
2 Arrays
12 Arrays
24 Arrays
4 Arrays*
25 mL
2 Arrays
12 Arrays
24 Arrays
4 Arrays*
25 mL
2 Arrays
12 Arrays
24 Arrays
4 Arrays*
25 mL
330520
330522
330523
330521
330529
330510
330512
330513
330511
330519
330500
330502
330503
330501
330509
PA-012
PA-012-12
PA-012-24
PA-012-8
PA-112
PA-011
PA-011-12
PA-011-24
PA-011-8
PA-111
PA-010
PA-010-12
PA-010-24
PA-010-8
PA-110
9. RT2 Profiler PCR ARRAY Performance
Reliable and Reproducible Pathway-focused Gene Expression Analysis
B
9.95
Fluorescence
RT2 Profiler PCR Arrays are used and trusted by thousands of research scientists
for pathway-focused gene expression analysis. Several factors, including the RT2
Primer Assay design algorithm, the proprietary control panel, and the strict
manufacturing and quality control procedures, ensure the outstanding
performance and reliability of our PCR Arrays. Each PCR Array and every qPCR
Primer Assay is wet-bench verified to guarantee their performance, with results
demonstrating several performance parameters illustrated here.
7.95
5.95
108
109
3.95
107
106
0.5
0
2
4
8
6
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle Number
Uniform PCR Amplification Efficiency
One prerequisite for PCR Array technology is that the amount of template product
doubles with every cycle. The more the assays deviate from this ideal, the error
in the fold change calculation (∆Ct) increases exponentially. Only with consistently high amplification efficiencies can PCR Arrays yield meaningful comparison of gene expression levels of all genes simultaneously. The unique combination of SABiosciences' proprietary primer design algorithm and rigorous testing
of every primer assay by hand guarantees the high performance of every primer
assay on the PCR Arrays.
Regardless of user or instrument used, the complete PCR Array System demonstrates strong correlations across technical replicates, lots, and instruments with
average correlation coefficients > 0.99 insuring reliable detection of differences
in expression between biological samples.
0.1
0
50
40
60
70
90
80
TM [ C]
99
120
PreAMP
100
Unamp
% Increase in Positive Call Rate
80
60
40
20
0
100
50
25
10
Input RNA [ng]
1
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
% Increase
Positive Call Rate
A key benefit of using pathway-focused PCR Arrays for gene expression analysis is
that genes that are over expressed can be measured as reliably as those that are
under expressed. The complete PCR Array System yields > 85% positive call with 25
ng - 5 µg RNA or >90% with as a little as 1 ng PreAMP RNA. The 8-log wide dynamic
range provided by real-time PCR is unparalleled when comparing a pathway-focused
gene panel of varying expression levels across a variety of samples.
Figure 2: PCR Arrays Detect as Little as 1 ng RNA. Different amounts of universal total
RNA were characterized using the Human Inflammatory Cytokines and Receptors PCR Array
(PAHS-011) with or without PreAMP. The percentage of detectable genes was calculated for each
RNA amount, with 1ng RNA analysis enabled with the new pathway-focused PreAMP technology.
A1
A2
Ct from User A
High Sensitivity and Wide Dynamic Range
A4
Universal total RNA was characterized for four chemokine and chemokine receptors using RT2 Primer
Assays, followed by a dissociation (melt) curve analysis. PCR Arrays specifically detect individual genes
despite the expression of related gene family members in the same RNA sample.
% Positive Call
B1
o
Figure 1: PCR Arrays Amplify A Single Gene-Specific Product in Every Reaction.
A
1
Superb Reproducibility
0.2
-0.1
101
102
Figure 3: PCR Arrays Detect RNA Across a Wide Dynamic Range. Ten-fold serial
dilutions of Human CHRNA5 were characterized with the respective RT2 qPCR Primer Assay.
A3
Signal [-d(RFU)/dT]
CXCL3
CXCR1 CXCR2 CXCL2 CXCL3
0.3
CXCR1
CXCR2
CXCL2
Agarose Gel
0.4
103
104
19
.5
Distinct Specificity
The complete PCR Array System , with high quality input RNA, is guaranteed to
yield single bands without primer dimers or other secondary products. The
proprietary primer design algorithm incorporates more than ten thermodynamic
and sequence alignment criteria, and our wet-bench verification provides
confidence that every real-time qPCR Assay accurately represents the expression
of the queried gene. Over 20,000 gene-specific RT2 PCR Primer Assays have been
designed and shipped to satisfied customers.
105
40
30
20
10
0
0
40
30
20
10
0
0
40
30
20
10
0
0
40
30
20
10
0
0
10
20
30
10
20
30
10
20
30
10
20
30
40
30
20
10
0
40 0
40
30
20
10
0
40 0
40
30
20
10
0
40 0
40
30
20
10
0
40 0
B2
10
20
30
10
20
30
10
20
30
10
20
30
40
30
20
10
0
40 0
40
30
20
10
0
40 0
40
30
20
10
0
40 0
40
30
20
10
0
40 0
B3
10
20
30
40
10
20
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40
10
20
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40
10
20
30
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10
0
0
40
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10
0
0
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0
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B4
10
20
30
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10
20
30
40
10
20
30
40
10
20
30
40
Ct from User B
Figure 4: PCR Arrays Yield Highly Reproducible Results. Four replicate sets of raw
threshold data (1-4) obtained by two different scientists (A & B) at two different times on Human Drug
Metabolism RT2 Profiler PCR Arrays are directly compared. The results demonstrate a high degree of
correlation (R2 > 0.990).
RT2 Profiler PCR Arrays: A Trusted & Reliable System
PCR Arrays have been used by thousands of researchers who have successfully
submitted and published their PCR Array results in very high impact journals,
including Science, PNAS, Cancer Research, the Nature & Cell family of journals,
and others (See Pages 24-27).
www.SABiosciences.com
7
10. RT2 Nano PreAMP cDNA Synthesis Technology
TM
Enabling the Analysis of 1 Nanogram of RNA with RT2 ProfilerTM PCR ARRAYS
2
TM
What Is the RT Nano PreAMP cDNA Synthesis Kit?
RT² Nano PreAMP cDNA Synthesis Kit and Primer Mixes are a breakthrough
technology enabling expression analysis starting from as little as 1 ng of total
RNA. It employs a proprietary preamplification process to faithfully increase the
amount of targeted cDNA for PCR Array analysis. This technology empowers RT²
Profiler PCR Arrays to accurately analyze nanogram levels of total RNA.
2
How RT Nano PreAMP cDNA Synthesis Kits Work
Two Simple Steps:
AAAAA
AAAAA
AAAAA
AAAAA
1. cDNA First Strand Synthesis
Samples that can NOW be characterized with real-time PCR Arrays include:
Laser Captured Microdissection Samples (LCM)
mRNA
This kit provides enough reagents for synthesizing first strand cDNA from 12
different samples of as little as 1 ng total RNA.
2. Preamplification of cDNA for Pathway Specific Genes
Fine Needle Aspiration Biopsies (FNAB)
Each first strand cDNA synthesis reaction can be amplified by 4 different
sets of PCR Array-specific Primer Mixes, allowing gene expression analysis
for one sample on as many as 4 different PCR Arrays.
Stem Cell Clusters or Embryoid Bodies
Flow Cytometry / Fluorescent-Activated Cell Sorting (FACS)
Combined with PCR Arrays, the RT² Nano PreAMP cDNA Synthesis Kit and Primer
Mixes extends the PCR Array System to accurately analyze a pathway-focused
set of genes with as little as 1 ng of total RNA.
∆Ct - PreAMP cDNA
Unbiased Amplification Process & Comparison of ∆Ct
Values Between Preamplified and Unamplified cDNA
RT 2 Nano PreAMP cDNA Synthesis Kit:
Proprietary kits include
optimized reagents for first strand cDNA synthesis and preamplification from only 1 ng
of total RNA.
RT 2 Nano PreAMP cDNA Synthesis Primer Mixes:
Ready-to-use
primer mixes for amplifying pathway-specific cDNA templates on corresponding RT2
Profiler PCR Arrays.
Benefits of RT2 Nano PreAMP cDNA Synthesis Technology
Robust Performance on Small Samples:
Arrays starting with as little as 1 ng of Total RNA.
Analyze up to 4 different PCR
Easy Workflow and Designed for Routine Use: Simple and quick
procedures with minimal hands-on time to preamplify target templates in under 2 hrs.
Superior Sensitivity: Maximally enhances the sensitivity of RT Profiler PCR
Arrays to analyze limited amounts of RNA.
2
100
Unamp
% Increase in Positive Call Rate
80
60
40
20
0
100
50
25
10
Input RNA [ng]
1
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Support@SABiosciences.com
5
10
15
Figure 2. Unbiased Amplification Process - Highly Comparable ∆Ct Values Between
Preamplified and Unamplified cDNA from Human Liver Tumor RNA. First strand cDNA
was synthesized from 5 ng of human liver tumor RNA. One-quarter of each RT product was used for
preamplification with the RT2 Nano PreAMP cDNA Synthesis Kit plus the Human Cancer PathwayFinderTM Nano PreAMP Primer Mix. Unamplified cDNA synthesized from 500 ng of the same liver tumor RNA
sample was used as the control. PreAMP amplified and unamplified cDNA samples were then analyzed
on the Human Cancer PathwayFinderTM PCR Array, and the threshold cycle values (Ct) were obtained. The
∆Ct value for each gene was calculated by subtracting the average Ct value of the five reference genes
(B2M, HPRT1, RPL13A, GAPDH, and ACTB) on the PCR Array from the Ct value of each gene of interest.
The dashed line represents the ideal slope of 1.0. The solid line shows a linear regression fit with the
R2 and slope indicated.
Product
(red) or without (blue) RT Nano preamplification. The unamplified and preamplified samples were then
analyzed on the Human Inflammatory Cytokines and Receptors PCR Array (PAHS-011), which contains 84
pathway-specific assays, plus controls, including 5 assays for housekeeping genes. Threshold cycle
values (Ct) were obtained and any genes with a Ct < 35 were considered to be present. Results indicate
that with Nano preamplification, a 33.7% increase in positive call rate is observed in samples with as
little as 1 ng RNA.
8
-5
2
Figure 1. RT2 Nano Preamplification Significantly Increases Sensitivity of Detection
from 1 ng RNA Samples. Different amounts of human universal RNA were converted to cDNA with
2
-5
RT Nano PreAMP Products
% Increase
Positive Call Rate
% Positive Call
PreAMP
R2 = 0.95
Slope = 1.01
5
∆Ct - Unamplified cDNA
Nano PreAMP Further Increases
the Sensitivity of PCR ARRAYS
120
15
10
RT Nano PreAMP cDNA Synthesis Kit
2
RT Nano PreAMP cDNA Synthesis Primer Mixes*
Human Cancer PathwayFinderTM
Human Apoptosis
Human Angiogenesis
Human Mesenchymal Stem Cell
Human Embryonic Stem Cell
Human Extracellular Matrix and Adhesion Molecules
Human Inflammatory Cytokines and Receptors
Mouse Inflammatory Cytokines and Receptors
Human Toll-like Receptor Signaling Pathway
Mouse Toll-like Receptor Signaling Pathway
2
RT Nano PreAMP Primer Mixes for All PCR Arrays
2
* PreAMP Primer Mixes for Custom PCR Arrays available
Catalog #
SABio #
330454
330241
330241
330241
330241
330241
330241
330241
330241
330241
330241
330241
Inquire
330454
PBH-033
PBH-012
PBH-024
PBH-082
PBH-081
PBH-013
PBH-011
PBM-011
PBH-018
PBM-018
Inquire
11. RT2 FFPE PreAMP cDNA Synthesis Technology
TM
Enabling the Analysis of FFPE Samples with RT2 Profiler PCR Arrays
RNA Extraction from
FFPE Samples
(330471)
The combination of a simplified Xylene-Free RNA extraction and a high-fidelity
amplification process maximizes recovery of RNA and microRNA (miRNA). RT²
Profiler PCR Arrays facilitate easy and reliable expression analysis of genes
associated with a biological pathway or a diseased state from FFPE samples.
First Strand cDNA
Synthesis and
Preamplification
(330461 +
330251:PFX-####)
Benefits of RT FFPE PreAMP PCR ARRAY System
Quick and Efficient: High quality and high-yield total RNA and miRNA isolation
from FFPE samples in 70 minutes
Superior Sensitivity:
Simple Xylene-Free procedure and robust performance
PCR ARRAY Detection
RT2 FFPE Preamplification Faithfully
Represents Biological Changes
30
.
Real-time PCR Detection
RT2 ProfilerTM PCR Array
-3.
0
30
25
Figure 1. Highly Comparable Gene Expression Fold Change Results between FFPE
Preamplified and Unamplified Samples. RNA extracted from FFPE spleen and intestine
samples were extracted using the RT2 FFPE RNA Extraction Kit and converted to cDNA with and without
preamplification. All four cDNAs were analyzed on the Human Cancer PathwayFinderTM PCR Array. The
∆∆Ct comparison and genes with raw Ct values lower than 33 in both unpreamplified spleen and
intestine samples are presented.
JUN
IFNB1
FFPE PreAMP ∆∆Ct
CDC25A
15
-5.
0
ANGPT1
-4.
0
TIMP3
20
TWIST1
-2.
0
35
TERT
3.
0
MET
2.
0
MMP1
1.
0
IFNA1
-1.
0
RT Only
RT + PreAMP
E2F1
-1.
0
RT2 SYBR®
Green
Master Mix
40
FGFR2
-2.
0
10
.
CDKN2A
-.
30
45
First-Strand cDNA Synthesis
and PreAMP Multiplex PCR
RT2 FFPE PreAMP Increases Detection of
Genes Previously Classified as “Absent”
CASP8
-.
40
20
.
Raw Ct
FFPE ∆∆Ct
-.
50
Nucleic Acid Extraction
2 HOURS
PreAMP protocol significantly enhances qRT-PCR
detection sensitivity for FFPE samples
y = 0.9749x - 0.0589
R2 = 0.9187
Proteinase K Digestion
Column Clean-up
2
Easy Workflow:
Cut FFPE Sections / Slides
(10 - 80 µm)
70 MINUTES
An innovative solution enabling the accurate qRT-PCR analysis of Formalin-fixed
Paraffin-embedded (FFPE) samples. The RT² FFPE PreAMP technology utilizes
multiplex tandem PCR to preamplify gene-specific cDNA with minimal bias. This
kit is intended for preamplification of first-strand cDNA from fragmented total
RNA from FFPE samples for gene expression analysis with RT² Profiler PCR
Arrays.
How the RT2 FFPE PreAMP PCR System Works
90 MINUTES
Gene Expression Analysis from FFPE Samples
Figure 2. Genes extracted from FFPE samples previously classified as “Absent” are
now detectable after RT2 FFPE Preamplification. RNA was extracted from FFPE spleen
sample (human) with the RT2 FFPE RNA Extraction Kit and reverse transcribed to cDNA using RT2 FFPE
preamplification (red bars) and without PreAMP (blue bars). Results of the Human Cancer PathwayFinder
PCR Array showed 55% of unpreamplified genes were virtually undetectable with no genes in the 10-20 Ct
range. Preamplified genes with Ct values > 30, shift into the reliably quantitative range (Ct = 10-30).
RT FFPE PreAMP & RNA Extraction Products
2
RT2 FFPE PreAMP Performance
Increased positive call rate from FFPE samples
Increased detection of genes previously classified as “Absent”
Unbiased amplification of preamplified genes
Faithful conservation of biological changes
Product
Catalog #
RT FFPE RNA Extraction Kit
2
RT FFPE PreAMP cDNA Synthesis Kit
2
RT FFPE PreAMP Primer Mixes for All PCR ARRAYS
2
RT FFPE PreAMP Primer Mixes for Custom PCR ARRAYS
2
RT Profiler PCR ARRAYS
2
RT SYBR® Green qPCR Master Mixes
330471
330461
330251
2
www.SABiosciences.com
9
12. Data Analysis
Quick and Easy Software
Service Core
Gene Expression Analysis Services
FREE Web-Based PCR ARRAY Data Analysis Software
Genomic & Gene Expression Analysis
This integrated web-based software package for the PCR Array System automatically performs all ΔΔCt based fold-change calculations from your uploaded raw
threshold cycle data. Simply providing the array's catalog number annotates the
results to the correct gene list. The web portal delivers results not only in a
tabular format but also in scatter, volcano, cluster-gram, and multi-group plots.
Perform any pair-wise comparison between groups of experimental replicates by
defining your own fold-change and statistical significance thresholds, or compare
all of the groups side-by-side. The web portal also helps you correctly interpret
the genomic DNA, reverse transcription efficiency, and positive PCR control well
data. Make your pathway-focused gene expression analysis quick and painless
with the PCR Array System and the PCR Array Data Analysis Suite.
SABiosciences provides genomic and expression analysis services that deliver
robust and reproducible results. The Service Core accepts and processes a
variety of biological samples and then leverages cutting-edge tools for either
pathway-focused or genome-wide analysis yielding superior results for scientists
in academic, government and industrial settings.
Available Analysis Services
DNA Service
Whole Genome
Simple: Just upload your data and define your parameters*
Convenient: No downloading or installation required
RNA Service
Illumina Genotyping
Illumina Expression Profiling
Methylation
Illumina DASL (fixed samples)
Methylation
Individual Gene / Locus
Methylation
qPCR - SYBR® Green
Cells
Cells
Tissue or Blood
Tissue or Blood
Fixed Tissue
Fixed Tissue
Small Sample
Small Sample
Publication-Ready Output: Export all results as FREE EXCEL files or
PNG image files
* EXCEL-based data analysis templates are available from our website.
INSTRUCTIONS
1
2
3
PCR Array
Pathway-focused
Sample Preparation
Upload your data in a simple EXCEL file format.
Define your housekeeping genes and experimental groups.
Choose an automatically generated data analysis result
Take a test run with pre-loaded sample data set today:
www.SABiosciences.com/pcrarraydataanalysis.php
OR
Join our next live webinar entitled: “PCR Array Data Analysis Tutorial” at:
www.SABiosciences.com/seminarlist.php
Volcano Plot
Scatter Plot
miRNA PCR Array
Nucleic Acid Isolation
Sample isolation and integrity are fundamental to the success of any biological
analysis. The Service Core provides qualified expertise in RNA and DNA
isolation from a variety of biological samples. Each sample is evaluated for
concentration and degradation to ensure successful future analysis.
• RNA / DNA Isolation from Cells, Tissue or Biofluids (USA only)
• Integrity/Degradation Checked for Every Sample
Whole Genome Analysis
LOG10 [p-Value]
LOG10 [Group 1]
LOG10 [Control Group]
Figure 1: The Scatter Plot Compares
Gene Expression Levels Between Two
Experimental Conditions. The graph plots
the log10 of normalized gene expression levels in a
control condition (x-axis) versus an experimental
condition (y-axis). Symbols outside the gray area
indicate fold-differences larger than a threshold
that you can define. The red symbols in the
upper-left corner readily identify up-regulated
genes, and the green symbols in the lower right
corner readily identify down-regulated genes.
10
The Service Core uses the Illumina Platform to provide whole genome expression
profiling, cytogenetic analysis, methylation and single nucleotide polymorphism
(SNP) genotyping. As an Illumina CSPro (certified service provider), scientists are
receiving results from a trained and tested scientific staff.
LOG2 [Group 1/Control Group]
Figure 2: The Volcano Plot Indicates
the Statistical Significance of Gene
Expression Changes. The x-axis plots the
log2 of the fold-differences, while the y-axis
plots their p-values based on student’s t-test of
your replicate raw Ct data. The red and green
symbols outside the gray area conveniently have
the same meaning as the Scatter Plot. Symbols
in the Volcano Plot above the blue line readily
identify fold-differences at least as statistically
significant as a threshold that you can define.
Support@SABiosciences.com
• Expression Profiling (Human, Mouse or Rat)
• SNP Genotyping & Copy Number Variation Analysis
• Whole Genome Methylation Analysis (Human)
Pathway-focused Analysis
Real-time PCR is a powerful tool for validating results from whole genome
expression profiling, methylation or pathway analysis. An experienced leader in
providing qPCR services, the Service Core delivers rapid and reproducible results.
• Custom PCR Array Analysis
• Pathway-focused PCR Array
• Methyl-Profiler PCR Array
13. QIAGEN® RNeasy® Kits
Purification of High-quality Total RNA from a Range of Biological Samples
A
RNeasy Kits are a proven technology for rapid and convenient purification of
®
high-quality RNA. Reproducible yields of intact RNA with high Agilent RIN (RNA
Integrity Number) values are obtained, ensuring reliable results in downstream
applications such as real-time RT-PCR. Kits are available for cells and easy-tolyse tissues as well as for more challenging samples, such as fiber-rich or fatty
tissues, fine needle aspirates, and cryosections.
Why Use RNeasy Kits?
QIAGEN QIAxcel®
Rel. FI Units [RFU, x1E+000]
What Are RNeasy Kits?
1799.4 nt
20
4628.7 nt
10
131.0 nt
10 min
5 min
B
Agilent Bioanalyzer
400
How Do RNeasy Kits Work?
Biological samples are first lysed in a lysis buffer that contains a guanidine salt,
which fully denatures RNases to prevent RNA degradation. RNA is then specifically bound to a silica membrane, either in an RNeasy spin column or the well of
an RNeasy 96 plate. Other cellular material is efficiently washed away using a
series of wash buffers before pure, intact RNA is eluted in RNase-free water.
300
FU
When purifying RNA, it is important to use a method that maintains RNA integrity
and removes contaminants. Degradation of RNA makes reliable analysis of gene
expression impossible, while the presence of contaminants in the purified RNA
can inhibit enzymes in downstream applications such as real-time RT-PCR and
microarray analysis. RNeasy Kits overcome these challenges through the
combination of a specialized lysis buffer and silica-membrane technology.
200
100
0
25
200 500 1000
2000
4000
Figure 1. Highly Intact RNA. RNA was purified from Jurkat cells using the RNeasy Mini Kit. The
purified RNA was analyzed on the [A] QIAxcel system (ratio of 28S to 18S rRNA: 1.55) and [B] Agilent
2100 Bioanalyzer (ratio of 28S to 18S rRNA: 1.7). A high RNA Integrity Number (RIN) of 9.6 was
obtained, indicating highly intact RNA.
QIAGEN RNeasy Kits*
Product
Catalog #
RNeasy Mini Kit (50)
For purification of RNA from cells & easy-to-lyse tissues
Sample
Lyse, homogenize,
and add ethanol
Bind total RNA to
RNeasy membrane
74104
RNeasy Fibrous Tissue Mini Kit (50)
For purification of RNA from fiber-rich tissues
RNeasy Procedure
74704
RNeasy Lipid Tissue Mini Kit (50)
For purification of RNA from all tissue types
74804
RNeasy 96 Kit (12)
For purification of RNA from cells in 96-well format
74182
QIAzol Lysis Reagent (200 mL)
For lysis of fatty & standard tissues before RNA isolation.
79306
* See also page 22 for RNeasy Protect Kits for stabilization and purification of RNA from tissues, cells,
2
saliva, and blood. See page 7 for for details on the RT FFPE RNA Extraction Kit.
Wash
RNeasy Fibrous Tissue Mini Kit
Elute in small volume
Ready-to-use RNA
The RNeasy Fibrous Tissue Mini Kit includes proteinase K
for removing abundant protein in fiber-rich tissue samples,
and RNeasy spin columns for purifying up to 100 µg of
high-quality RNA. Fibrous tissue samples can be
conveniently stabilized using RNAlater RNA Stabilization
Reagent or Allprotect Tissue Reagent, and efficiently
disrupted using a TissueRuptor or TissueLyser system.
www.SABiosciences.com
11
14. QIAGEN® Products for Sample Disruption
Fast and Effective Disruption of Biological Samples at a Range of Throughputs
What Is Sample Disruption?
QIAGEN Sample Disruption Products
Effective disruption and homogenization of a biological sample is an absolute
requirement for all RNA purification procedures. Disruption releases the RNA
contained in a sample, while homogenization reduces sample viscosity to
facilitate subsequent RNA purification.
Product
QIAshredder (50)
For homogenization of cell lysates
®
Catalog #
79654
TissueRuptor
For disruption of individual samples
9001271
TissueRuptor Disposable Probes (25)
Disposable probes for use with the
TissueRuptor
990890
TissueLyser LT
For disruption of up to 12 samples
85600
TissueLyser LT Adapter, 12-tube
Adapter for use with the TissueLyser LT
69980
300
TissueLyser II
For disruption of up to 48 or 192 samples
85300
200
TissueLyser Adapter Set 2 x 24
Adapter set for use with the TissueLyser II;
holds 48 tubes
69982
TissueLyser Adapter Set 2 x 96
Adapter set for use with the TissueLyser II;
holds 192 tubes
69984
Stainless Steel Beads, 5mm (200)
69989
Stainless Steel Beads, 7mm (200)
69990
Tungsten Carbide Beads, 3mm (200)
69997
TissueLyser Single-Bead Dispenser, 5mm
69965
The QIAshredder is a biopolymer-shredding system in a spin-column format. Cell
lysate is applied to a QIAshredder spin column, which is then briefly centrifuged
to homogenize the lysate.
TissueLyser Single-Bead Dispenser, 7mm
69967
TissueLyser 3 mm Bead Dispenser, 96-Well
69973
The TissueRuptor is a handheld device that provides simultaneous disruption and
homogenization using TissueRuptor Disposable Probes, which contain a blade
that rotates at very high speeds. As the probes are both disposable and transparent, the risk of cross-contamination is minimized and the sample disruption
process can be visually monitored. Use of disposable probes also saves time as
there is no need to clean the same probe after disrupting each sample.
TissueLyser 5 mm Bead Dispenser, 96-Well
69975
Why Use QIAGEN Sample Disruption Products?
QIAGEN provides a range of technologies for disruption and homogenization —
from QIAshredder spin columns for fast and simple homogenization of cell lysates
to TissueRuptor and TissueLyser systems for mechanical disruption and homogenization of tougher tissue samples at a range of throughputs. TissueRuptor and
TissueLyser systems deliver fast and effective disruption, and replace tedious
and time-consuming methods such as disruption using a mortar and pestle.
RNA Concentration [ng/µL]
400
TissueLyser LT
TissueLyser II
100
0
Skin
Heart
Lung
Brain
Figure 1. Effective Tissue Disruption. Various rat tissues were disrupted using the TissueLyser
LT or TissueLyser II. RNA was purified from 20 mg samples on the QIAcube using the RNeasy Fibrous
Tissue Mini Kit (skin, heart, and lung) or RNeasy Lipid Tissue Mini Kit (brain). RNA was eluted in a
volume of 50 µL, and concentration was determined using a spectrophotometer.
How Do QIAGEN Sample Disruption Products Work?
TissueLyser instruments are bead mills that simultaneously disrupt and homogenize samples through high-speed shaking with grinding beads in plastic tubes.
Using an adapter that holds several tubes, the instruments disrupt multiple
samples at the same time — up to 12 samples with the TissueLyser LT, and up to
48 or 192 samples with the TissueLyser II.
12
Support@SABiosciences.com
The TissueLyser LT
The TissueLyser LT is a small bead mill which provides fast,
effective disruption of up to 12 samples at the same time.
Simultaneous disruption and homogenization is achieved
through high-speed shaking of samples in 2 mL microcentrifuge tubes with stainless steel or glass beads.
15. QIAGEN® Products for RNA Stabilization
Convenient and Immediate Stabilization of RNA in a Range of Biological Samples
What Is RNA Stabilization?
QIAGEN RNA Stabilization Products*
Once a biological sample is harvested, its RNA becomes extremely unstable. The
RNA is degraded by RNases, and gene induction or downregulation triggered by
sample manipulation will also occur. Immediate stabilization of cellular RNA to
preserve mRNA levels is critical for accurate gene expression analysis.
RNA stabilization is usually achieved by rapidly freezing samples in liquid
nitrogen or on dry ice. However, the use of such chemicals is hazardous, and care
should be taken to avoid thawing of samples prior to sample disruption and RNA
purification.
Why Use QIAGEN RNA Stabilization Products?
QIAGEN provides a broad range of reagents for convenient stabilization of RNA
in cells, tissues, blood, and saliva at room temperature. The use of hazardous
liquid nitrogen or dry ice to freeze samples is avoided. Samples are simply
submerged in the reagents to immediately preserve the gene expression profile,
and can then be conveniently handled and transported at ambient temperature
prior to RNA purification. For further convenience, stabilization reagents are also
available as part of QIAGEN kits for RNA purification.
Fold Change in Transcript Level
Allprotect Tissue Reagent (100 mL)
For stabilization of RNA, DNA, and protein in tissues
®
AllPrep DNA/RNA Mini Kit (50)
For simultaneous purification of DNA and RNA
from cells and tissues
®
Catalog #
76405
80204
RNAlater RNA Stabilization Reagent (250 mL)
For stabilization of RNA in tissues
76106
RNeasy Protect Mini Kit (50)
For stabilization of RNA in tissues and purification of RNA
74124
®
RNAprotect Cell Reagent (250 mL)
For stabilization of RNA in cells
76526
RNeasy Protect Cell Mini Kit (50)
For stabilization of RNA in cells and purification of RNA
74624
RNeasy Protect Saliva Mini Kit (50)
For stabilization of RNA in saliva and purification of RNA
74324
RNAprotect Animal Blood Tubes (50 x 100 µL)
For collection of 100 µL animal blood with RNA
stabilization
76544
RNAprotect Animal Blood Tubes (50 x 500 µL)
For collection of 500 µL animal blood with RNA
stabilization
76554
RNeasy Protect Animal Blood Kit (50)
For purification of RNA from blood collected in
RNAprotect Animal Blood Tubes
73224
miRNeasy Protect Animal Blood Kit (50)
For purification of RNA, including miRNA, from blood
collected in RNAprotect Animal Blood Tubes
c-fos Transcript in Lung
100
217304
80
60
40
20
0
2 Hours
4 Hours
Allprotect
24 Hours
2 Hours
4 Hours
PBS
24 Hours
Madh7 Transcript in Intestine
10
Fold Change in Transcript Level
Product
1
* For collection of human blood with RNA stabilization and purification, use the PAXgene Blood RNA
System. For collection of tissues with preservation of histomorphology and nucleic acids and nucleic
acid purification, use the PAXgene Tissue System. For details, visit www.PreAnalytiX.com.
0.1
0.01
0.001
2 Hours
4 Hours
Allprotect
24 Hours
2 Hours
4 Hours
PBS
24 Hours
Figure 1. Effective RNA Stabilization. Rat tissues were stored at 25ºC for 2–24 hours in
Allprotect Reagent or PBS prior to real-time RT-PCR analysis. Transcript levels relative to those in liquid
nitrogen stabilized tissues were calculated. Changes in transcript levels were prevented by Allprotect
Reagent.
Allprotect Tissue Reagent
Allprotect Tissue Reagent provides immediate stabilization
of DNA, RNA, and protein in tissue samples at room
temperature. It is ideal for use prior to simultaneous
purification of DNA, RNA, and protein using AllPrep Kits.
www.SABiosciences.com
13
16. APOPTOSIS
BIOMARKERS
CANCER
CELL CYCLE
PATHWAY-POWERED
RT2 PROFILERTM PCR ARRAYS
• Technology Overview
(page 2)
• Over 100 Pathways Available
(page 28)
Breast Cancer and Estrogen
Receptor Signaling
Apoptosis
Autophagy
Cancer PathwayFinder
TM
Cell Cycle
DNA Damage Signaling
Pathway
Endothelial Cell Biology
• Start With as Little as 1 ng of RNA
(page 8)
• Analyze FFPE Samples with PCR Arrays
(page 9)
• Over 1,000 Peer-reviewed Publications
• Custom PCR Arrays Available for
Human, Mouse, Rat, Rhesus Macaque
and Drosophila
Heat Shock Proteins
NFκB Signaling Pathway
Oxidative Stress and
Antioxidant Defense
p53 Signaling Pathway
PI3K-AKT Signaling
Pathway
Stress and Toxicity
PathwayFinder
Angiogenesis
Apoptosis
Cell Surface Markers
Apoptosis
Autophagy
Breast Cancer and Estrogen
Receptor Signaling
Cancer PathwayFinder
Cancer Drug Resistance
and Metabolism
DNA Damage Signaling
Pathway
Dendritic and Antigen
Presenting Cell
Epigenetic Chromatin
Modification Enzymes
Epigenetic Chromatin
Remodeling Factors
Epithelial to Mesenchymal
Transition (EMT)
Extracellular Matrix and
Adhesion Molecules
Glucose Metabolism
Hematopoietic Stem Cells
and Hematopoiesis
Homeobox (HOX) Genes
TNF Ligand and Receptor
Mesenchymal Stem Cell
Unfolded Protein Response
Stem Cell
T-cell and B-cell Activation
• To Learn More, Please Visit the PCR
Product Web Page:
Th1-Th2-Th3
• Accurate Detection of DNA Methylation
at CpG Islands Without Bisulfite
(page 20)
RT2 MicroRNA PCR ARRAYS
• Regulation of Gene Transcription and
Translation (page 16)
• Human, Mouse & Rat
(Sanger miRBase 14)
ChampionChIPTM PCR ARRAYS
• Analyze DNA-Protein (histone)
Interactions (page 19)
14
Cell Cycle
Epithelial to Mesenchymal
Transition (EMT)
DNA Damage Signaling
Pathway
MAP Kinase Signaling
Pathway
Epithelial to Mesenchymal
Transition (EMT)
Neurogenesis and Neural
Stem Cell
EGF / PDGF Signaling
Pathway
NFκB Signaling Pathway
MAP Kinase Signaling
Pathway
PI3K-AKT Signaling
Pathway
p53 Signaling Pathway
Protein Phosphatases
PI3K-AKT Signaling Pathway
TGFβ BMP Signaling
Pathway
Tumor Metastasis
www.SABiosciences.com/RTPCR.php
Methyl-ProfilerTM PCR ARRAYS
Cancer PathwayFinder
Cell Cycle
p53 Signaling Pathway
Protein Phosphatases
Signal Transduction
PathwayFinder
Transcription Factors
Ubiquitination Pathway
Wnt Signaling Pathway
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
Tumor Suppressor Genes*
Tumor Suppressor Genes*
Cancer
Cancer
Cancer
Cancer
Cell Differentiation and
Development
Cell Differentiation and
Development
miFinderTM
Cell Differentiation and
Development
miFinderTM
miFinderTM
Genome v2.0*
Genome v2.0*
Oncogenes and Tumor
Suppressor Genes
Oncogenes and Tumor
Suppressor Genes
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
(Signature & Complete Panels)
Support@SABiosciences.com
(Signature & Complete Panels)
(Signature & Complete Panels)
Genome v2.0*
(Signature & Complete Panels)
miFinderTM
Genome v2.0*
Oncogenes and Tumor
Suppressor Genes
T Helper Cell Differentiation
17. CYTOKINES /
INFLAMMATION
NEUROSCIENCE
STEM CELL /
SIGNAL
TRANSDUCTION DEVELOPMENT
Angiogenic Growth Factors
& Angiogenesis Inhibitors
Alzheimer's Disease
cAMP / Ca2+ Signaling
PathwayFinderTM
Dendritic and Antigen
Presenting Cell
Cancer Drug Resistance
and Metabolism
Inflammatory Cytokines
and Receptors
Atherosclerosis
Embryonic Stem Cells
Embryonic Stem Cells
Cancer PathwayFinderTM
Inflammatory Response
and Autoimmunity
Common Cytokine
GPCR Signaling
PathwayFinder
EGF / PDGF Signaling
Pathway
Hedgehog Signaling
Pathway
DNA Damage Signaling
Pathway
Embryonic Stem Cells
Heat Shock Proteins
Endothelial Cell Biology
Hedgehog Signaling
Pathway
Hematopoietic Stem Cells
and Hematopoiesis
Drug Metabolism
Interferon and Receptor
Chemokines & Receptors
Common Cytokine
Interferon α, β Response
JAK / STAT Signaling
Pathway
NFκB Signaling Pathway
T Cell Anergy & Immune
Tolerance
T-cell and B-cell Activation
TGFβ BMP Signaling
Pathway
Th17 for Autoimmunity and
Inflammation
Th1-Th2-Th3
Toll-Like Receptor
Signaling Pathway
ECM /
ADHESION
Chemokines and Receptors
Extracellular Matrix and
Adhesion Molecules
Apoptosis
Hypoxia Signaling Pathway
GPCR Signaling
PathwayFinderTM
Hedgehog Signaling
Pathway
Homeobox (HOX) Genes
Mesenchymal Stem Cell
MAP Kinase Signaling
Pathway
Neurogenesis and Neural
Stem Cell
Mesenchymal Stem Cell
Neuroscience Ion Channels
and Transporters
Osteogenesis
TGFβ BMP Signaling
Pathway
Tumor Metastasis
TNF Ligand and Receptor
Neurotransmitter Receptors
and Regulators
Neurotrophin and
Receptors
Nitric Oxide Signaling
Pathway
Notch Signaling Pathway
Hepatotoxicology
Notch Signaling Pathway
Nuclear Receptors and
Coregulators
GPCR Signaling
PathwayFinder
Neurotrophin & Receptors
NFκB Signaling Pathway
NFκB Signaling Pathway
Drug Transporters
Neurogenesis and Neural
Stem Cell
MAP Kinase Signaling
Pathway
Drug Metabolism:
Phase II Enzymes
Mesenchymal Stem Cell
JAK / STAT Signaling
Pathway
Drug Metabolism:
Phase I Enzymes
Lipoprotein Signaling and
Cholesterol Metabolism
Insulin Signaling Pathway
Glycosylation
Lipoprotein Signaling &
Cholesterol Metabolism
Osteogenesis
PI3K-AKT Signaling
Pathway
Stem Cell Signaling
T-cell and B-cell Activation
Signal Transduction
PathwayFinder
Terminal Differentiation
Marker
TGFβ BMP Signaling
Pathway
Transcription Factors
Inflammatory Response
T Cell Activation
Cytokine Production
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
Inflammatory Response
(Signature & Complete Panels)
Cancer
Genome v2.0
miFinder
Immunopathology
Breast, Colon, Gastric,
Liver, Lung, & Prostate
Cancer
Stem Cell Transcription
Factors
(Signature & Complete Panels)
Homeobox* (HOX)
Polycomb* (PcG)
Mitochondria
Nephrotoxicity
Oxidative Stress and
Antioxidant Defense
Stress and Toxicity
PathwayFinder
Inflammatory Response
T Cell Activation
Cytokine Production
Cancer
Cancer
miFinderTM
Genome v2.0*
Cell Differentiation and
Development
Cell Differentiation and
Development
Genome v2.0*
miFinderTM
miFinderTM
Genome v2.0*
Genome v2.0*
Oncogenes and Tumor
Suppressor Genes
Stem Cell Transcription
Factors
Genome v2.0*
TM
Inflammation
T Helper Cell Differentiation
Wnt Signaling Pathway
Molecular Toxicology 384HT
miFinderTM
miFinderTM
Inflammatory Response
TGFβ BMP Signaling
Pathway
Wnt Signaling Pathway
TNF Ligand and Receptor
TOXICOLOGY /
DRUG ADME
Stem Cell Transcription
Factors
Inflammatory Response
T Helper Cell Differentiation
www.SABiosciences.com
15
18. RT2 MicroRNA PCR ARRAYS
Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA
How miRNA PCR ARRAYS Work
As Easy as a Real-time PCR Experiment
MicroRNAs are endogenous single-stranded RNA molecules 19-25 nucleotides in
length, synthesized in a regulated manner from larger RNA molecules. Many
miRNA sequences have been found in a variety of species [miRBase Release 14:
>760 miRNAs in humans, >575 in mice, and >370 in rats].
1. Convert miRNA to cDNA via Universal Tailing
and Reverse Transcription.
miRNA
Sample 1
Why Use SABiosciences miRNA PCR ARRAYS?
miRNA
Sample2
miRNA
5’
Identification of miRNAs that potentially regulate your genes of interest is
possible with our powerful yet simple bioinformatic algorithm at:
www.SABiosciences.com/miRNAsearch.php
3.
Functional Studies:
The function of individual miRNAs can be identified via miRNA over expression or suppression of miRNA function.
16
Support@SABiosciences.com
1 hr
Universal Priming
AAAAAA
5’
TTTTTT
Universal RT
Primer
Reverse Transcription
AAAAAA
cDNA
5’
TTTTTT
2 hr
cDNA
5’
TTTTTT
3’
qPCR using miRNASpecific Primer and
Universal qPCR Primer
4. Data Analysis.
Specificity
120
Single Nucleotide Mismatch Specificity
Targeted Template
80
72.2
20
0
0.54
hsa-miR-99a
0.07
hsa-miR-100
miR-99a
miR-100
hsa-miR-99a
21.76
O F
F
40
O T R E
N A G T
60
SABiosciences Assay
B
Off-Target Template
100
O F T R E
F
A G T
A
Expression Analysis:
Bioinformatic Prediction:
Polyadenylation
3’
3. Run in Your Real-Time PCR Instrument.
The best technology for determining the expression of miRNA is the
SABiosciences miRNA PCR Array System.
2.
5’
3’
O T R E
N A G T
1.
Analyze 96 - 384
miRNAs in Each
PCR Array
O T R E
N A G T
MicroRNA represents a new layer of regulation in endogenous gene transcription
and translation. Since there are hundreds of miRNAs in each species, with each
miRNA potentially having hundreds of targets, the majority of genes may be
subject to regulation by one or more miRNAs. miRNAs are already being considered as cancer biomarkers, and their importance is being realized in a variety of
other research areas, such as differentiation, neurobiology and immunology.
There are three major ways to start studying miRNA:
AAAAAA
Poly(A) tail
2. Add cDNA to RT2 qPCR Master Mix.
Aliquot Mixture Across PCR Array.
Sensitivity: As little as 0.5 µg total RNA needed
Multi-Sequence Flexibility: Analyze up to 384 sequences simultaneously
Simplicity: As easy as a real-time PCR Array experiment
Why Study miRNA?
Single Tube
Reaction
5’
3’
2
The RT miRNA PCR Array accurately analyzes the expression of up to 96 or 384
microRNA sequences simultaneously on ANY real-time PCR instrument. SABiosciences' patent-pending miRNA technology integrates a universal-tailing and
reverse transcription reaction specific for miRNA with accurate expression level
measurement of distinct miRNA sequences that may differ by a single nucleotide
base. RT2 miRNA PCR Arrays are the most specific and sensitive technology for
analyzing genome-wide miRNA expression.
3’
5’
Relative Detection [% Correct]
Detecting every miRNA across the entire genome in a specific and sensitive way
is a very technologically challenging task. Many miRNA family members and
otherwise distinct miRNA species have very similar sequences. Moreover, other
RNA species such as snRNA, tRNA, mRNA, and rRNA can cause non-specific
amplification, making the specific analysis of mature miRNA even more problematic. SABiosciences' proprietary miRNA detection technologies enable uniformly
high PCR amplification efficiencies, allowing simultaneous detection of miRNA
under uniform cycling conditions.
O T R E
N A G T
What are miRNAs?
hsa-miR-100
Competitor Assay
AACCCGUAGAUCCGAUCUUGUG
AACCCGUAGAUCCGAACUUGUG
Figure 1: miRNA qPCR Assays Distinguish Single Nucleotide Mismatches. RT2 miRNA
PCR Assays and a competitor’s assays for miR-99a and miR-100 were used to detect both corresponding
synthetic templates, whose sequences differ by only one nucleotide (B). Relative detection of the
off-target template is calculated as a percentage of the correct template detection (A). RT2 miRNA PCR
Assays’ proprietary primer design specifically discriminates closely related sequences better than
competing assays.
19.
20. QIAGEN® miRNeasy Kits
Purification of Total RNA from a Range of Biological Samples
A
What Are miRNeasy Kits?
miRNeasy Kits are a special adaptation of proven RNeasy technology, allowing
purification of total RNA longer than approximately 18 nucleotides. The purified
RNA includes large RNAs, such as mRNA and rRNA, as well as small RNAs, such
as microRNA (miRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA
(snoRNA), and small nuclear RNA (snRNA).
High-throughput
Ct Value
32
Why Use miRNeasy Kits?
Threshold Cycles
25
22
12
1x107
1x106
1x103
1x102
2 µg
200 ng
Low-throughput
32
Ct Value
Consistent Results
1x104
1x105
Cell Number
B
The miRNeasy Mini Kit and miRNeasy 96 Kit allow efficient purification of
miRNA from all types of cells and tissues. Effective lysis, even with difficult-tolyse tissues, and removal of contaminants by organic-phase extraction is
®
achieved using QIAzol Lysis Reagent. RNA is then purified in spin-column
format (miRNeasy Mini Kit) or in 96-well format (miRNeasy 96 Kit).
30
27
17
Copurification of large and small RNAs allows real-time RT-PCR detection of both
mRNA and miRNA using, for example, the miScript PCR System and RT2 Profiler
PCR Arrays. For certain applications where large RNAs need to be removed,
supplementary protocols are available for purification of an RNA fraction rich in
small RNAs.
How Do miRNeasy Kits Work?
Copurification
miRNA-enriched Fraction
Copurification
miRNA-enriched Fraction
27
22
17
QIAcube
Manual
12
20
20 mg
2 mg
200 µg
20 µg
Amount of Tissue
15
Figure 2. Effective Purification from a Range of Starting Amounts. Total RNA was
purified from [A] 102–107 Jurkat cells using the miRNeasy 96 Kit or [B] a dilution series of rat lung tissue
homogenate from 20 mg to 200 ng using the miRNeasy Mini Kit. miRNA-enriched fractions (<200
nucleotides) were also isolated from the same samples. Purified RNA was used as a template in
quantitative, real-time RT-PCR assays for the miRNA miR-16.
10
5
0
miR-16
miR-25
Figure 1. Reliable miRNA Detection. Rat kidney (25 mg) was stabilized in RNAlater RNA
Stabilization Reagent and disrupted using the TissueLyser II. Total RNA (with miRNA) was purified using
the miRNeasy Mini Kit, either manually or on the QIAcube®. Real-time RT-PCR using the miScript PCR
System was carried out to detect miR-16 and miR-25.
miRNeasy Mini Kit
The miRNeasy Mini Kit enables purification of total RNA
which includes RNA from approximately 18 nucleotides (nt)
upwards from all types of animal tissues and cells, including
difficult-to-lyse tissues. The miRNeasy Mini Kit is used for
low-throughput RNA purification using spin columns.
QIAGEN miRNeasy Kits*
Product
miRNeasy Mini Kit (50)
For purification of RNA, including miRNA,
from cells and tissues
217004
miRNeasy 96 Kit (4)
For purification of RNA, including miRNA,
from cells and tissues in 96-well format
217061
* See also page 22 for stabilization and purification of RNA, including miRNA, from blood.
2
for for details on the RT FFPE RNA Extraction Kit that also recovers miRNA.
18
Support@SABiosciences.com
Catalog #
See page 7
21. ChampionChIP qPCR System
TM
40%
35%
30%
25%
The ChampionChIP One-Day Kit simplifies the usual two- to five-day ChIP protocol down to a manageable six to eight hours. Its crosslink reversal step is much
faster and less tedious than conventional methods, and its DNA purification step
yields a larger quantity and higher quality ChIP DNA than other one-day kits.
20%
15%
10%
5%
0%
CDKN1A Gene Region (kb Relative to TSS)
ChampionChIP antibodies for modified histones (H3Ac, H3K4me2, H3K27me3) or control IgG were used
for precipitating chromatin from one million HeLa cells. Each ChIP DNA fraction was analyzed with a
ChampionChIP Tiling Array representing 30 one-kb tile intervals across the genomic sequence of the
CDKN1A gene. The results obtained from three independent experiments are consistent with active
transcription of the CDKN1A gene.
8
30 min
30 min
40 min
Optional: Quickly Evaluate Fragmentation Size
2. Immunoprecipitation
Pre-Clear
Anti-TF or -Histone Antibody & Control IgG
Save
Input
Protein A Beads
Fraction
Wash
3. ChIP and Input Fraction DNA Isolation
Reverse Cross-Linking and Elution
DNA Spin-Column Purification
B. Real-Time PCR
C. Data Analysis (ChIP PCR Array Analysis Software)
50 min
1-2 hr
1 hr
30 min
30 min
10 min
2 hr
15 min
Fold Change
7
A. Chromatin Immunoprecipitation (ChIP)
1. ChIP-Ready Chromatin Preparation
Fix and Harvest Cells
Sonicate Chromatin
6
5
4
The ChampionChIP System includes a simplified high-performance One-Day ChIP Kit, ChIP-Grade
Antibodies, real-time PCR primers, and a FREE ChIP PCR Array Data Analysis Suite.
Percent of DNA Input [%]
Euchromatin and Heterochromatin Markers
H3K4me2
H3K27me3
H3K9me3
Control IgG
6
4
2
0
A549
HepG2
PC3
0
p53 Binding
CDKN1A
mRNA Expression
Figure 4: Treatment with 5-Fluorouracil Increases CDKN1A Gene Expression and p53
Binding in Cell Lines Expressing Wild-Type But Not Mutant p53. Triplicate samples from
A549, HepG2, and PC3 cells were treated with 5-FU (300 µM, 6 h), and either subjected to ChIP with an
anti-p53 antibody followed by qPCR analysis of the CDKN1A-2kb p53 binding site, or harvested for RNA
to analyze CDKN1A expression by real-time RT-PCR. The results of both assays are expressed as the
fold-increase upon 5-FU treatment.
ChampionChIP qPCR System
Catalog #
Human
Mouse
ChampionChIP PCR Arrays
Stem Cell Transcription Genes
Oncogene & Tumor Suppressor Genes
T Helper Cell Differentiation
Inflammatory Responses
ChampionChIP One-Day Kit
ChampionChIP Antibody Kits
Human RNA Polymerase II, p53
Histones (H3Ac, H4Ac, H3Kme2, etc.)
330454
330454
330454
330454
330454
334471
GH-501
GH-502
GH-503
GH-504
GA-101
GM-501
GM-502
GM-503
GM-504
GA-101
334481
Inquire
Inquire
ChampionChIP qPCR Primers
High Specificity
8
Transcription Factor Binding and mRNA Expression Analysis
3
2
1
Figure 1: The Entire ChampionChIP System Protocol Can Be Completed in a Single Day.
10
H3Ac
H3K4me2
H3K27me3
Control IgG
Figure 3: Differential Histone Modification Tiling Across the Sequence of Any Gene.
How the ChampionChIPTM System Works
12
Differential Histone Modification Across Any Gene
+10
+9
+8
+7
+6
+5
+4
+3
+2
+1
-1
-2
-3
-4
-5
-6
-7
-8
-9
-10
-11
-12
-13
-14
-15
-16
-17
-18
-19
-20
The ChampionChIPTM System provides the first complete platform for the analysis
of in vivo protein-DNA interactions using Chromatin Immunoprecipitation (ChIP)
and real-time PCR (qPCR) detection. With validated high-quality ChIP-grade
antibodies and PCR primers for any genes’ promoter region, a simple and robust
one-day preparation assay quickly delivers reliable and biologically relevant
results. ChIP qPCR is a powerful and versatile method for the analysis of chromatin DNA bound by transcription factors, co-regulators, modified histones, chromatin remodeling proteins, or other nuclear factors from live cells. However, the
tedious process and variable results have limited many researchers’ ability to
adopt this technique to study dynamic protein-DNA interactions in native
chromatin environments. The ChampionChIPTM System, yields the most reliable
ChIP assay results with qPCR precision in just a single day.
Percent of DNA Input [%]
Reliable Chromatin IP with Real-Time PCR Precision Achieved in Only ONE DAY!
334001
Inquire
Inquire
Custom ChampionChIP qPCR Arrays
Inquire
Inquire
Inquire
Product
Any Promoter Region for Human, Mouse or Rat
ALDOA
MYO-D
Genomic Loci
SAT2
TF Binding Sites or Promoter Tiling
330520
RT SYBR Green w/ ROX Master Mix
2
330510
RT SYBR Green w/ Fluorescein Master Mix
2
330500
RT SYBR Green Only Master Mix
ChampionChIP PCR Array Data Analysis Software
2
Figure 2. The ChampionChIP System Readily and Correctly Identifies Different Euchromatin and Heterochromatin Loci. ChampionChIP antibodies against modified histones (H3K4me2,
H3K27me3, H3K9me3) or control IgG were used for precipitating chromatin from HeLa cells. Each ChIP
DNA fraction was analyzed by real-time PCR using primers specific for the ALDOA, MYO-D, and SAT2
loci to calculate percentages of co-precipitating DNA relative to input.
www.SABiosciences.com
FREE
19
22. Methyl-Profiler PCR ARRAY System
TM
Simple, Fast and Reliable DNA Methylation Analysis
The Methyl-ProfilerTM DNA Methylation PCR Array System is an innovative technology enabling fast and accurate detection of DNA methylation status at CpG
islands. This technology complements the bisulfite-based methods with simple
and simultaneous selective restriction digests of either methylated or unmethylated DNA, and takes advantage of the quantitative power of real-time PCR. The
PCR Array format reveals the DNA methylation status of gene panels related to
diseases or pathways. The individual primer pairs allow analysis of the DNA
methylation status of any human or mouse gene. The reliability and simplicity of
the procedure makes this technology ideal for profiling DNA methylation and
biomarkers of stem cell growth and differentiation, cancer, and other human
diseases.
How DNA Methylation PCR ARRAYS Work
1. DNA Digestion.
Mix DNA +
Digestion Buffer
Split into 4 Fractions
Why Use the Methyl-ProfilerTM PCR ARRAY System?
Simple, Fast and Reliable:
No bisulfite conversion and ready-to-use.
Disease- or Pathway-Focused Gene Sets:
Simultaneously detect DNA methylation of 24 or 96 genes.
Genome-wide Coverage:
Primers to detect methylation of your favorite genes.
The Methyl-Profiler PCR Array System relies on the differential cleavage of
target sequences by two different restriction endonucleases whose activities
require either the presence or absence of methylated cytosines in their respective recognition sequences. As real-time PCR quantifies the relative amount of
intact DNA remaining after each enzyme digestion, the methylation status of
individual genes and the methylation profile across a gene panel are reliably and
easily calculated. The high yield of DNA from the restriction digests and PCR
amplification allow the analysis of smaller, more heterogeneous samples.
Results Comparable to Bisulfite Sequencing
Add Enzyme:
Mock
Sensitive
Dependent
Methylation Sensitive
-
+
-
+
Methylation Dependent
-
-
+
+
37 C (6 hr - overnight)
RT SYBR Green qPCR Master Mix
2
®
Mock
Sensitive
Dependent
Double
% Total Input DNA
Hypermethylated
Intermediately Methylated
Unmethylated
Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
Gene 6
The human genome contains many long hypermethylated stretches of CpG
dinucleotide-rich sequences. In this sea of CpG methylation, unmethylated
CpG-rich sequences, known as “CpG islands”, are found in the promoters of most
transcriptionally active genes. These normal patterns of DNA methylation are
perturbed in cancer cells, where specific tumor suppressor genes (TSG) become
hypermethylated, causing their expression to be silenced. Since every tumor type
has a unique “methylation profile”, or panel of hypermethylated genes, the
analysis of TSG hypermethylation has become very important for basic cancer
research, clinical diagnostics, and therapeutic applications.
The Methyl-Profiler PCR Array System fulfills the need to rapidly and simultaneously determine the methylation status of more genes in more samples in a
higher-throughput fashion. The current time- and labor-intensive methodologies
require bisulfite conversion of unmethylated cytosines to uracil followed by
either sequence analysis or PCR using primers sensitive to the resulting base
conversion. Bisulfite conversion is not only tedious but is also inefficient and
damages DNA. The resulting low yields of DNA make bisulfite-based methods
unsuitable for the analysis of small samples.
20
90
Support@SABiosciences.com
Hypermethylated
Intermediately Methylated
Unmethylated
80
70
60
50
40
30
20
10
0
3. Data Analysis.
100
80
60
40
20
0
Percent of Input DNA [%]
Double
o
2. Real-Time PCR.
100
Bisulfite
Sequencing
MethylProfiler
MCF7
Bisulfite
Sequencing
MethylProfiler
SKBR3
Bisulfite
Sequencing
MethylProfiler
MDA-MB-231
Figure 1: Methyl-Profiler PCR Assays Yield Results Consistent with Bisulfite Sequencing.
The methylation status of the cadherin 1 gene (CDH1) was determined using bisulfite sequencing and
Methyl-Profiler PCR Assays in three breast cancer cell lines known to have very different CDH1
methylation patterns.
To validate the accuracy of the Methyl-Profiler PCR Array System, its results were
compared with those generated by bisulfite sequencing, the gold standard for
DNA methylation analysis. The methylation status for both the CDH1 (Figure 1)
and CDH13 (data not shown) genes observed in three different breast cancer cell
lines by the two methods match very closely. Real-time PCR characterization of
methylation-dependent and methylation-sensitive restriction enzyme digestions
directly quantifies unmethylated and hypermethylated genomic DNA, respectively. The results indicate that the sensitivity and specificity of the MethylProfiler PCR Array System rivals bisulfite sequencing, suggesting that it can
provide an alternative to more tedious bisulfite PCR analysis methods.
Make your DNA Methylation analysis as quick and painless as possible with the
Methyl-Profiler DNA Methylation PCR System and EXCEL-based data analysis.
Download our FREE EXCEL Data Analysis Software:
www.SABiosciences.com/dna_methylation.php
23. Methyl-ProfilerTM PCR ARRAY System
PCR ARRAY Guide v4.0
Novel Breast Cancer Biomarker Discovery
100
Hypermethylated
Intermediately Methylated
Unmethylated
80
70
60
50
40
30
20
10
0
100
93.75
87.5
75
50
33.33
12.5
6.25
0
and normal blood genomic DNA (encoding hypermethylated and unmethylated HIC1, respectively) were
mixed in different ratios. Using Human HIC1 Methyl-Profiler qPCR Primers, the percentage of hypermethylated HIC1 relative to total promoter DNA in each mixture was detectable down to 6.25 percent.
Primary tumors are typically very heterogeneous, containing a mixture of both
cancerous and noncancerous cells. Therefore, reliable tumor characterization
requires detecting smaller amounts of hypermethylated DNA diluted in an
unmethylated background. Methyl-Profiler PCR Assays have the sensitivity
required to detect hypermethylated DNA from breast cancer cells even when
they represent only 6.25 % of the total cell population (Figure 2).
Applications
MLH1
ATM
APC
CHFR
CAV1
CRBP1
FHIT
BRCA1
CDKN2A
CDKN1A
SFN
p14ARF
PYCARD
GADD45A
ZMYND10
GSTP1
CDH1
CALCA
MGMT
ABCB1
CDH13
WT1
p73
HIC1
% Hypermethylated DNA
30%
40%
50%
60%
70%
>80%
Blood
MCF7
SKBR3
24 Signature Breast Cancer Tumor Suppressor Genes
Breast Cancer Biomarker Validation
20%
20%
30%
40%
50%
60%
70%
Normal MDA- MDA- UACC812 MDA- SKBR3 SKBR3 MCF7 MCF7
MB-415 MB-231
MB-435
Figure 2: Methyl-Profiler PCR Assays Detect Hypermethylation in Heterogeneous
Samples Containing As Little As Five Percent Tumor DNA. SKBR3 breast cancer cell line
10%
10%
>80%
Percent SKBR3 Genomic DNA [%]
<10%
79 Transcription Factor Genes
<10%
90
% Hypermethylated DNA
Percent of Total Input DNA [%]
Sensitivity Comparable to Bisulfite Sequencing
MDA-MB-231
Cell Lines
Figure 3: Methyl-Profiler PCR Arrays Validate Breast Cancer Gene Methylation
Status in Breast Cancer Cell Lines. Heat map comparison of the hypermethylation status of 24
genes in the genomic DNA of three breast cancer cell lines and blood genomic DNA as determined by
Human Breast Cancer Signature Panel DNA Methylation PCR Arrays.
To demonstrate that Methyl-Profiler PCR Arrays can validate methylation
biomarkers, we first scanned published results to design a cataloged PCR Array
representing a signature panel of the 24 most frequently methylated genes in
human breast tumors. We then analyzed the methylation profile of this gene
panel in three different breast cancer cell lines (Figure 3). The results further
strengthen the correlation of these biomarkers with breast cancer.
Cell Lines
Figure 4: Methyl-Profiler PCR Arrays Discover New Candidate Breast Cancer DNA
Methylation Biomarkers. Heat map comparison of the hypermethylation status of a panel of 79
transcription factor genes in six breast cancer cell lines and a normal epithelial cell line as determined
with Custom DNA Methylation PCR Arrays.
To demonstrate that Methyl-Profiler PCR Arrays can also discover new biomarkers,
we arranged a custom array containing a panel of candidate transcription factor
genes, whose methylation status had not been previously associated with breast
cancer (Figure 4). We found that breast cancer cell lines also hypermethylate this
gene panel, potentially providing a new discovery source for cancer biomarkers.
The Methyl-Profiler DNA Methylation PCR Array System is ideally suited to
genomic DNA hypermethylation analysis for both basic research applications and
clinical biomarker development. The simple two-step procedure is considerably
faster and easier than current bisulfite sequencing and bisulfite PCR methods,
and yields closely matching results with equivalent sensitivity.
Methyl-Profiler
TM
DNA Methylation PCR ARRAYS
Product*
Breast Cancer
Gastric Cancer
Liver Cancer
Lung Cancer
Prostate Cancer
Colon Cancer
Human Stem Cell Transcription Factors
Inflammatory Response
T Cell Activation
Cytokine Production
Tumor Suppressor Genes (TSG)
Homeobox Genes (HOX)
Polycomb Genes (PcG)
Custom Methyl-Profiler PCR Arrays
Catalog #
Human
Mouse
335211
335211
335211
335211
335211
335211
335211
335211
335211
335211
335211
335211
335211
MeAH-011
MeAH-021
MeAH-031
MeAH-041
MeAH-051
MeAH-061
MeAH-511
MeaH-521
MeaH-531
MeAH-541
MeAH-551
MeAH-561
MeAH-571
Inquire
MeAM-011
MeAM-021
MeAM-031
MeAM-041
MeAM-051
MeAM-061
MeAM-511
MeAM-521
MeAM-531
MeAM-541
MeAM-551
MeAM-561
MeAM-571
Inquire
* Methyl-Profiler PCR Arrays are available in Signature Panels (24 genes) & Comprehensive Panels (96 genes).
PCR ARRAY Accessories
Pack Size
Methyl-Profiler qPCR Assays
200 Reactions
Methyl-Profiler Enzyme Kit
12 Samples
2
RT SYBR Green Master Mixes (see page 6)
2 Arrays
Methyl-Profiler PCR Array Data Analysis Software
www.SABiosciences.com
Catalog #
335001
335451
Various
FREE
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24. QIAGEN® DNeasy® Blood & Tissue Kits
Purification of Total DNA from Cells, Tissues & Blood
What Are DNeasy Blood & Tissue Kits?
DNeasy Blood & Tissue Kits provide fast and easy silica-based DNA purification
in convenient spin-column and 96-well-plate formats. Most samples can be
directly lysed with proteinase K, eliminating the need for mechanical disruption
and reducing hands-on time. Optimized protocols for specific sample types
provide reproducible purification of high-quality DNA for life science, genotyping,
and veterinary pathogen research applications.
Why Use DNeasy Blood & Tissue Kits?
DNeasy Blood & Tissue Kits simplify purification of DNA from a wide range of
sample types, including animal species commonly encountered in life science,
veterinary, and genotyping applications. The efficient DNeasy Blood & Tissue
procedure enables high yields of total DNA from animal blood and tissue
samples.
Typical Yields Using DNeasy Blood & Tissue Kits
Source
Amount
DNA [µg]
Mammalian Blood
Bird Blood
HeLa Cells
Liver
Brain
Kidney
Spleen
Mouse Tail
Rat Tail
Pig Ear
Horse Hair
Fish Fin
Fish Spawn (mackerel)
100 µL
5 µL
2 x 106
25 mg
25 mg
25 mg
10 mg
1.2 cm (tip)
0.6 cm (tip)
25 mg
10 hairs
20 mg
10 mg
3-6
9-40
15-25
10-30
15-30
15-30
5-30
10-25
20-40
10-30
2-4
10-20
5-10
QIAGEN DNeasy Blood & Tissue Kits
How Do DNeasy Blood & Tissue Kits Work?
Product
DNeasy Blood & Tissue Kits use reliable silica-membrane technology, in
convenient spin-column or 96-well formats. This technology ensures fast and
reproducible DNA purification, eliminating the need for organic extraction and
alcohol precipitation.
Catalog #
69581
69582
69506
69504
DNeasy 96 Blood & Tissue Kit (4) 4 x 96 DNA minipreps
DNeasy 96 Blood & Tissue Kit (12) 12 x 96 DNA minipreps
DNeasy Blood & Tissue Kit (250) 250 DNA minipreps
DNeasy Blood & Tissue Kit (50) 50 DNA minipreps
QIAGEN® QIAamp® DNA Mini Kits
Purification of Genomic, Mitochondrial, Bacterial, Parasitic, or Viral DNA
What are QIAamp DNA Mini Kits?
QIAamp DNA Mini Kits provide silica-membrane-based nucleic acid purification
from tissues, swabs, CSF, blood, body fluids, or washed cells from urine. The
spin-column procedure does not require mechanical homogenization, so total
hands-on preparation time is only 20 minutes.
Why use QIAamp DNA Mini Kits?
QIAamp DNA technology yields genomic, mitochondrial, bacterial, parasite, or viral
DNA from human tissue samples ready to use in PCR and blotting procedures.
DNA purified using the QIAamp DNA Mini Kit is sized up to 50 kb. DNA of this
length denatures completely and has the highest amplification efficiency.
How do QIAamp DNA Mini Kits work?
QIAamp DNA Mini Kits use fast spin-column or vacuum procedures. No phenolchloroform extraction is required. DNA binds specifically to the QIAamp silica-gel
membrane while contaminants pass through. PCR inhibitors such as divalent
cations and proteins are completely removed in two efficient wash steps, leaving
purified DNA to be eluted in either water or a buffer provided with the kit.
22
Support@SABiosciences.com
Typical Yields Using QIAamp DNA Mini Kits
Sample
Blood
Buffy Coat
Cells
Liver
Brain
Lung
Heart
Kidney
Spleen
Amount
Total Nucleic Acids [µg]*
DNA [µg]†
200 µL
200 µL
107
25 mg
25 mg
25 mg
25 mg
25 mg
10 mg
4-12
25-50
40-60
60-115
35-60
25-45
15-40
40-85
25-45
4-12
25-50
30-40
10-30
15-30
5-10
5-10
15-30
5-30
* Nucleic acids obtained without RNase treatment.
† DNA obtained with RNase treatment.
QIAGEN QIAamp DNA Mini Kits
Product
Catalog #
QIAamp DNA Mini Kit (250) For 250 DNA preps
51306
QIAamp DNA Mini Kit (50) For 50 DNA preps
51304
25. PreAnalytiX® PAXgene® DNA System
Collection & Stabilization of Blood or Tissue Samples & Subsequent DNA Purification
What Is the PAXgene DNA System?
A
For blood, the PAXgene Blood DNA System is an integrated and standardized
system for collection and stabilization of whole blood specimens and subsequent
purification of genomic DNA. The system uses PAXgene Blood DNA Tubes for
blood collection and stabilization, and the PAXgene Blood DNA Kit for
subsequent DNA purification. For tissue, the PAXgene Tissue DNA System
enables molecular and traditional pathology testing from the same specimen,
providing a superior alternative to traditional tissue fixation methods. PAXgene
Tissue Containers are used for collection, stabilization, and storage of tissue
specimens, while preserving tissue morphology. The PAXgene Tissue DNA Kit
allows subsequent purification of DNA.
Why Use the PAXgene DNA System?
The PAXgene Blood DNA System provides blood collection and purification in one
system, easy sample transport after collection, and high-quality,
high-molecular–weight DNA. The PAXgene Tissue DNA System provides an
integrated system for fixation, stabilization, and purification. It both preserves
tissue morphology and provides high-quality DNA.
How Does the PAXgene DNA System Work?
PAXgene Blood DNA Tubes are used for collection under vacuum of 8.5 mL whole
blood. These tubes contain a proprietary blend of reagents that both prevents
blood coagulation and stabilizes white blood cells. For DNA isolation, the blood
is transferred to processing tubes filled with cell lysis buffer and the solution is
mixed to lyse red and white blood cells. Cell nuclei and mitochondria are pelleted
by centrifugation, washed, and resuspended in digestion buffer. Protein contaminants are removed by incubation with a protease. DNA is precipitated in
isopropanol, washed in 70% ethanol, dried, and resuspended in resuspension
buffer. For tissue, samples are placed into PAXgene Tissue Containers. These
are dual-chamber containers prefilled with 2 reagents: PAXgene Tissue Fix which
rapidly penetrates and fixes the tissue and PAXgene Tissue Stabilizer which
stops fixation and stabilizes tissue. DNA purification is performed using the
PAXgene Tissue DNA Kit which uses silica-based technology in a spin-column
format.
M
M
48.5 kb
B
M
M
145.5 kb
48.5 kb
Figure 1. High Quality and High Molecular Weight of Genomic DNA.
Genomic DNA
was isolated from 8 blood donors using the PAXgene Blood DNA System. [A] Agarose gel analysis;
[B] pulsed-field gel electrophoresis for enhanced separation of high-molecular–weight genomic DNA;
[M] markers.
PreAnalytiX PAXgene DNA Kits
Product
Catalog #
761133
PAXgene Tissue Containers (10)
For 10 tissue samples
PAXgene Tissue Containers are dual-cavity containers prefilled with 2 reagents.
PAXgene Tissue Fix rapidly penetrates and fixes the tissue, preserving tissue
morphology. Fixation is comparable to formalin fixation, but without the destructive
nucleic acid crosslinking and degradation. After fixation, the tissue is transferred to
PAXgene Tissue Stabilizer in the same container. Nucleic acids and morphology of the
sample are stable up to 7 days at room temperature, for longer periods at 2-8°C, or
even at -20°C. Stabilized samples can be embedded in paraffin for histological studies.
PAXgene Tissue Kits provide efficient subsequent purification of RNA, miRNA, and/or
DNA from the same sample.
Inquire
PAXgene Blood DNA Kit (25)
For 25 DNA preparations
PAXgene Tissue Containers
PAXgene Blood DNA Tubes (100)
For collection of 100 samples
765112
PAXgene Tissue DNA Kit (50)
For 50 DNA preparations
767134
PAXgene Blood DNA Tubes
The PAXgene Blood DNA system consists of PAXgene
Blood DNA Tubes, for blood collection and stabilization,
and the PAXgene Blood DNA Kit, for DNA purification in
a single-tube procedure.
www.SABiosciences.com
23
26. RT2 Profiler PCR ARRAY Publications
Selected Research Papers Citing RT2 Profiler PCR ARRAYS*
References Listed by Pathway-Focused PCR Arrays
ANGIOGENESIS
APPLICATION:
CANCER BIOLOGY
Brant KA, Fabisiak JP. Nickel and the microbial toxin, MALP-2, stimulate proangiogenic
mediators from human lung fibroblasts via a HIF-1alpha and COX-2-mediated pathway.
Toxicol Sci. 2009 Jan;107(1):227-37.
Liu Z, Kobayashi K, van Dinther M, van Heiningen SH, Valdimarsdottir G, van Laar T,
Scharpfenecker M, Lowik CW, Goumans MJ, Ten Dijke P, Pardali E. VEGF and inhibitors of
TGFbeta type-I receptor kinase synergistically promote J Cell Sci. 2009 Sep 15;122(Pt
18):3294-302.
McElroy MK, Kaushal S, Tran Cao HS, Moossa AR, Talamini MA, Hoffman RM, Bouvet M.
Upregulation of thrombospondin-1 and angiogenesis in an aggressive human Mol Cancer
Ther. 2009 Jul;8(7):1779-86.
APOPTOSIS
Bose RN, Maurmann L, Mishur RJ, Yasui L, Gupta S, Grayburn WS, Hofstetter H, Milton T.
Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum-phosphato
complexes towards human ovarian cancer cells. Proc Natl Acad Sci U S A. 2008 Nov
25;105(47):18314-9.
Uetani T, Nakayama H, Okayama H, Okura T, Higaki J, Inoue H, Higashiyama S. Insufficiency
of proHB-EGF shedding enhances hypoxic cell death in H9c2 cardiomyoblasts via the
activation of Caspase-3 and c-JUN N-terminal kinase. J Biol Chem. 2009 Feb 4.
Bok K, Prikhodko VG, Green KY, Sosnovtsev SV. Apoptosis in Murine Norovirus Infected
RAW264.7 Cells is Associated with Survivin Downregulation. J Virol. 2009 Feb 11.
Mhyre AJ, Marcondes AM, Spaulding EY, Deeg HJ. Stroma-dependent apoptosis in clonal
hematopoietic precursors correlates with expression of PYCARD. Blood. 2009 Jan
15;113(3):649-58.
Pru JK, Kaneko-Tarui T, Jurisicova A, Kashiwagi A, Selesniemi K, Tilly JL. Induction of
proapoptotic gene expression and recruitment of p53 herald ovarian Reprod Sci. 2009
Apr;16(4):347-56.
BREAST CANCER & ESTROGEN RECEPTOR SIGNALING
Cooper C, Guo J, Yan Y, Chooniedass-Kothari S, Hube F, Hamedani MK, Murphy LC, Myal Y,
Leygue E. Increasing the relative expression of endogenous non-coding Steroid Receptor
RNA Nucleic Acids Res. 2009 Jul;37(13):4518-31.
Adams BD, Cowee DM, White BA. The role of miR-206 in the epidermal growth factor (EGF)
induced repression of Mol Endocrinol. 2009 Aug;23(8):1215-30.
TM
CANCER PATHWAYFINDER
Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK, Stodieck LS, Ferguson VL, Pecaut
MJ. Spaceflight effects on T lymphocyte distribution, function and gene expression. J Appl
Physiol. 2009 Jan;106(1):194-202.
Das KK, Bajpai M, Kong Y, Liu J, Geng X, Das KM. Mesalamine suppresses the expression of
TC22, a novel tropomyosin isoform Mol Pharmacol. 2009 Jul;76(1):183-91.
* For a complete list of publications, please visit: www.SABiosciences.com/support_publication.php
24
Support@SABiosciences.com
Figure: PIK3CA mRNA Expression in Multiple Lung Cancer Cell Lines. PIK3CA mRNA expression
was compared among cell lines having different features, such as PIK3CA alterations or mutations of
other genes involved in the EGFR signaling pathway. PIK3CA mRNA expression levels were expressed
relative to the mean levels in six HBEC cell lines. PIK3CA mRNA expression in PIK3CA gain or EGFR
mutant cell lines were significantly increased compared with that of wild-type cell lines. However,
PIK3CA mutant lines do not express increased mRNA levels. Horizonatal bars indicate mean values. The
Kruskal-Wallis test with Dunn’s multiple comparison test was used to determine significance.
Yamamoto, H., et al. Cancer Research 2008; 68: 6913-6921.
CELL CYCLE
Chen S, Sims GP, Chen XX, Gu YY, Chen S, Lipsky PE. Modulatory effects of
1,25-dihydroxyvitamin D3 on human B cell differentiation. J Immunol. 2007 Aug
1;179(3):1634-47.
Lu SY, Sontag DP, Detillieux KA, Cattini PA. FGF-16 is released from neonatal cardiac
myocytes and alters growth-related signaling: a possible role in postnatal development.
Am J Physiol Cell Physiol. 2008 May;294(5):C1242-9.
Higgins S, Wong SH, Richner M, Rowe CL, Newgreen DF, Werther GA, Russo VC.
Fibroblast growth factor 2 reactivates G1 checkpoint in SK-N-MC cells via Endocrinology.
2009 Sep;150(9):4044-55.
CHEMOKINES & RECEPTORS
Miselis NR, Wu ZJ, Van Rooijen N, Kane AB Targeting tumor-associated macrophages in an
orthotopic murine model of diffuse malignant mesothelioma. Mol Cancer Ther. 2008
Apr;7(4):788-99.
Lim J, Derrick SC, Kolibab K, Yang AL, Porcelli S, Jacobs WR, Morris SL. Early pulmonary
cytokine and chemokine responses in mice immunized with three different vaccines against
Mycobacterium tuberculosis determined by PCR array. Clin Vaccine Immunol. 2009
Jan;16(1):122-6.
Fukami N, Ramachandran S, Saini D, Walter M, Chapman W, Patterson GA, Mohanakumar
T. Antibodies to MHC class I induce autoimmunity: role in the pathogenesis of chronic
rejection. J Immunol. 2009 Jan 1;182(1):309-18.
Cheung KP, Yang E, Goldrath AW. Memory-like CD8+ T cells generated during homeostatic
proliferation defer to J Immunol. 2009 Sep 1;183(5):3364-72.
Sundararaj KP, Samuvel DJ, Li Y, Sanders JJ, Lopes-Virella MF, Huang Y. Interleukin-6
released from fibroblasts is essential for up-regulation of matrix J Biol Chem. 2009 May
15;284(20):13714-24.
27. PUBLICATIONS: PCR ARRAYS
COMMON CYTOKINES
Silver RF, Walrath J, Lee H, Jacobson BA, Horton H, Bowman MR, Nocka K, Sypek JP.
Human Alveolar Macrophage Gene Responses to Mycobacterium tuberculosis Strains
H37Ra and H37Rv. Am J Respir Cell Mol Biol. 2008 Sep 11.
Nacu N, Luzina IG, Highsmith K, Lockatell V, Pochetuhen K, Cooper ZA, Gillmeister MP, Todd
NW, Atamas SP Macrophages produce TGF-beta-induced (beta-ig-h3) following ingestion
of apoptotic cells and regulate MMP14 levels and collagen turnover in fibroblasts J
Immunol. 2008 Apr 1;180(7):5036-44.
Campeau PM, Rafei M, Boivin MN, Sun Y, Grabowski GA, Galipeau J. Characterization of
Gaucher disease bone marrow mesenchymal stromal cells reveals Blood. 2009 Oct
8;114(15):3181-90.
Shinoda Y, Ogata N, Higashikawa A, Manabe I, Shindo T, Yamada T, Kugimiya F, Ikeda T,
Kawamura N, Kawasaki Y, Tsushima K, Takeda N, Nagai R, Hoshi K, Nakamura K, Chung UI,
Kawaguchi H. Kruppel-like factor 5 causes cartilage degradation through transactivation of
matrix metalloproteinase 9. J Biol Chem. 2008 Jul 10.
Myskiw C, Arsenio J, van Bruggen R, Deschambault Y, Cao J. Vaccinia virus E3 suppresses
expression of diverse cytokines through inhibition J Virol. 2009 Jul;83(13):6757-68.
Lucio-Eterovic AK, Piao Y, de Groot JF. Mediators of glioblastoma resistance and invasion during
antivascular endothelial Clin Cancer Res. 2009 Jul 15;15(14):4589-99.
CUSTOM PCR ARRAYS
Kuhn AR, Schlauch K, Lao R, Halayko AJ, Gerthoffer WT, Singer CA. MicroRNA Expression in
Human Airway Smooth Muscle Cells: Role of miR-25 in Am J Respir Cell Mol Biol. 2009 Jun 18.
Huang J, Chen K, Huang J, Gong W, Dunlop NM, Howard OM, Bian X, Gao Y, Wang JM.
Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell
differentiation. Carcinogenesis. 2009 Feb;30(2):348-55.
ENDOTHELIAL CELL BIOLOGY
Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, Zoghbi HY. MeCP2, a Key Contributor to Neurological Disease, Activates and Represses Transcription Science. 2008 May
30;320(5880):1224-9.
Peduto L, Dulauroy S, Lochner M, Spath GF, Morales MA, Cumano A, Eberl G. Inflammation
recapitulates the ontogeny of lymphoid stromal cells. J Immunol. 2009 May 1;182(9):5789-99.
Ianzini F, Kosmacek EA, Nelson ES, Napoli E, Erenpreisa J, Kalejs M, Mackey MA.
Activation of meiosis-specific genes is associated with depolyploidization of Cancer Res.
2009 Mar 15;69(6):2296-304.
Schreiber TH, Deyev VV, Rosenblatt JD, Podack ER. Tumor-induced suppression of CTL expansion
and subjugation by gp96-Ig Cancer Res. 2009 Mar 1;69(5):2026-33.
DiMeo TA, Anderson K, Phadke P, Fan C, Perou CM, Naber S, Kuperwasser C. A novel lung
metastasis signature links Wnt signaling with cancer cell Cancer Res. 2009 Jul
1;69(13):5364-73.
DNA DAMAGE
Hoffman AE, Zheng T, Ba Y, Zhu Y. The circadian gene NPAS2, a putative tumor suppressor,
is involved in DNA damage response. Mol Cancer Res. 2008 Sep;6(9):1461-8.
Chesnokova V, Wong C, Zonis S, Gruszka A, Wawrowsky K, Ren SG, Benshlomo A, Yu R.
Diminished pancreatic {beta} cell mass in securin-null mice is caused by {beta} cell
apoptosis and senescence. Endocrinology. 2009 Feb 12.
Li Z, Suzuki Y, Huang M, Cao F, Xie X, Connolly AJ, Yang PC, Wu JC Comparison of Reporter
Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and
Differentiated Endothelial Cells in Living Subjects. Stem Cells. 2008 Apr;26(4):864-73.
Asada S, Takahashi T, Isodono K, Adachi A, Imoto H, Ogata T, Ueyama T, Matsubara H, Oh H.
Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells
to apoptosis. Am J Physiol Heart Circ Physiol. 2008 Dec;295(6):H2512-21.
GROWTH FACTORS
Stirling DP, Liu S, Kubes P, Yong VW Depletion of Ly6G/Gr-1 leukocytes after spinal cord
injury in mice alters wound healing and worsens neurological outcome. J Neurosci. 2009
Jan 21;29(3):753-64.
HYPOXIA SIGNALING
Wendler CC, Amatya S, McClaskey C, Ghatpande S, Fredholm BB, Rivkees SA. A1
adenosine receptors play an essential role in protecting the embryo against hypoxia. Proc
Natl Acad Sci U S A. 2007 Jun 5;104(23):9697-702.
Mueller BR, Bale TL. Sex-specific programming of offspring emotionality after stress early
in pregnancy. J Neurosci. 2008 Sep 3;28(36):9055-65.
APPLICATION:
INFLAMMATORY CYTOKINES & RECEPTORS
Santisteban M, Reiman JM, Asiedu MK, Behrens MD, Nassar A, Kalli KR, Haluska P, Ingle
JN, Hartmann LC, Manjili MH, Radisky DC, Ferrone S, Knutson KL. Immune-induced
epithelial to mesenchymal transition in vivo generates breast Cancer Res. 2009 Apr
1;69(7):2887-95.
DRUG METABOLISM: PHASE I ENZYMES
Ning B, Dial S, Sun Y, Wang J, Yang J, Guo L Systematic and simultaneous gene profiling of
84 drug-metabolizing genes in primary human hepatocytes J Biomol Screen. 2008
Mar;13(3):194-201.
EXTRACELLULAR MATRIX & ADHESION MOLECULES
Johansen LD, Naumanen T, Knudsen A, Westerlund N, Gromova I, Junttila M, Nielsen C,
Bottzauw T, Tolkovsky A, Westermarck J, Coffey ET, Jaattela M, Kallunki T. IKAP localizes to
membrane ruffles with filamin A and regulates actin cytoskeleton organization and cell
migration. J Cell Sci. 2008 Mar 15;121(Pt 6):854-64.
Figure: Lineage-dependent Differences in Expression of Treg Functional Genes. Quantitative
PCR analysis of selected genes related to Treg function in CD4+CD25+ cells from Mossi, Fulani, and
European donors infected with Plasmodium falciparum (malaria). CD4+CD25+ cells were isolated from 12
Mossi (red bars) and 12 Fulani (blue bars) donors included in the study and 10 European donors (gray
bars), and lysed to obtain total RNA. Equal amounts of RNA (50 ng) from each donor were reverse-transscribed and amplified in duplicate in RT2 Custom PCR Arrays to simultaneously examine the mRNA levels
of nine selected genes related to Treg activity using PPIA, GAPDH, and ACTB as housekeeping genes
(HKG). Data was normalized to the mean values of the HKG, and a relative amount of RNA was
calculated using the 2-∆C method.
t
Torcia MG, Santarlasci V,. et al. Proc Natl Acad Sci U S A. 2008 Jan 15;105:646-651.
www.SABiosciences.com
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