Sh rna 2013

SureSilencing™ shRNA Plasmids
Knock Down Your Favorite Genes
with Ease and Confidence with shRNA
Wei Cao, Ph.D.
Wei.Cao@QIAGEN.com

Technical Support:
Tel:
1-888-503-3187
Email: support@SABiosciences.com

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.

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International customers: sabio@qiagen.com
Webinar related questions: qiawebinars@qiagen.com

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-1-

Sample & Assay Technologies

Topics will be Covered
Topic I:
RNAi High Throughput Screening Applications, Challenges
and Solutions; May 6 , 1pm Eastern Time
https://www2.gotomeeting.com/register/786104298

Topic II (Today):
1

RNAi Introduction and Challenges

2

Solutions: SureSilencing shRNA Plasmid

3

Key Points to Ensure Successful RNAi

4

Application Examples

-2-


RNA Interference Introduction
What are siRNAs?
siRNAs are 21–23nt (nucleotide)
dsRNA duplexes with symmetric
2–3nt 3' overhangs and 5'phosphate and 3'-hydroxyl groups

How does it work?
Dicer delivers the siRNAs to a
group of proteins called the RISC
(RNA-Inducing Silencing
Complex)
siRNA duplex unwinds
Once unwound, the single-stranded
antisense strand guides RISC to
mRNA that has a complementary
sequence

-3-


RNAi Strategy: siRNA & shRNA
siRNA
Origin
Biogenesis

shRNA

Synthetic oligonucleotides
Introduced into cell by
transfection
Transient

Gene silencing
effect
Effect on protein Decrease protein levels
production
Quick transient knockdown,
not for long term knocking
down; not for enrichment
Applications

QIAGEN’s
Solution

FlexiTube siRNA
FlexiPlate siRNA

Plasmids or vector based
Synthesized in cell; enters RNAi pathway
Non-transient, long term effect
Decrease protein levels
Transient or stable transfection;
Build stable cell lines for renewable source
of gene knockdown;
Transfer shRNA between different vectors;
Inheritable silencing.

SureSilencing shRNA Plasmids

The choice of which one to use depends on the question under
investigation, the factors such as cell type, time demand, and the need for
transient or stable knockdown.
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Why Knockdown the Expression of a Gene?

RNAi

Gene Function Studies

Pathway Interrogation

Target Identification and Validation

Biomarker and Drug Target Discovery

-5-


RNAi Challenges
RNAi Knockdown Effectiveness
Differences exist between: Knockdown efficiencies advertised by
companies & observed by researchers

RNAi Specificity, Off-Target effect (OTE)
1. Sequence-specific OTEs
Mismatches between the siRNA guide strand and the complementary target
mRNA sequence, ‘seed region’
siRNAs function like microRNAs

2. Non-sequence-specific OTEs
Lipid-mediated response - cellular response to RNAi toxicity
Immune responses to RNAi, such as induction of Interferon pathway
RISC-dependent off-target effects

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Solutions @ QIAGEN


-7-


Available for every gene in the Human, Mouse, and Rat genome
Guaranteed >70% Knockdown by at least 2 different shRNAs
Power discovered by thousands of scientists worldwide
More than 200 publications in a broad range of research fields

Cancer
Stem Cell
Immunology
Neuroscience
Signal Transduction
Cell Differentiation
Cardiovascular Disease
Infectious Diseases (HIV, HCV)

Check website:

http://www.sabiosciences.com/support_publication.php

Customer’s success story:

http://www.sabiosciences.com/RNAipublication.php

-8-


SureSilencing shRNA – How it works

A vector is introduced into
cells and utilizes the U1
promoter to ensure that the
shRNA is always expressed
Dicer cleaves the shRNA
into siRNA.
The siRNA gene silencing
mechanism is followed.

-9-


SureSilencing shRNA Plasmids – Mechanism
U1 promoter transcribes
a moderate amount of shRNA

The ampicillin resistance
marker and bacterial
origin of replication permit
amplification for a
lifetime supply of plasmid

FACS enrichment

GFP

Antibiotic-resistance markers: Stable cell line development

Hyg

Neo

Puro

Enrich or Select: 4 Markers: GFP, Nyomycin, Hygromycin, and Puromycin
Multiple Designs: 4 Designs for each gene – each sequence targets different region
- 10 -


The Best shRNA Design Algorithm

Download White Paper “Did Your RNAi Experiment Work?!”
http://www.sabiosciences.com/validaternai.pdf
- 11 -


The Best RNAi Design Algorithm
Ensure Efficacy: Filter many chemical & sequence properties of siRNA known to be
important for activity
Length, between 19bps ~ 30bps
GC Content, between 32%~55%
Thermostability bias at 5’-end of antisense strand
Avoid tandem repeats and palindromes:
• no internal repeated sequences of length >=4;
• no GC stretch of length >=8;
• no repeats of AAA, UUU, GGG or CCC;
• no internal palindrome sequences of length >=5;

Ensure Specificity with Smith-Waterman sequence alignment algorithm, “Better than
BLAST”
Experimentally Validated shRNA Plasmids
2 of 4 successful designs per gene IS an Enforceable Guarantee!

Zhou H, Zeng X, Wang Y and Seyfarth BR. A Three-Phase Algorithm for Computer Aided siRNA Design.
Informatica.2006 30:357-364.
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Validation of SureSilencing shRNAs
Experimental Validation of shRNA Plasmids
329 Designs tested, 221 are successful: 67.2 % (>2/3);
86 Genes tested, 74 are successful: 86.0 % (>4/5);
Original publication by The RNAi Consortium (TRC) reports only 31-38 % (~1/3)
success rate using the same definition of success

Designs
Tested

Successful
Designs

Success
Rate (%)

Genes
Tested

Successful
Genes

Success
Rate (%)

SABio’s Set

329

221

67.2

86

74

86.0

The RNAi
Consortium

2561, 5422

971, 172

381, 312

53

40

75.5

1. RootRoot DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM. “Genome-scale loss-of-function screening with a lentiviral
RNAi library.” Nat Methods. 2006 Sep;3(9):715-9.
2. Moffat J, Grueneberg DA, Yang X, et. al. “A lentiviral RNAi library for human and mouse genes applied to an arrayed viral
high-content screen.” Cell. 2006 Mar 24;124(6):1283-98.

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SureSilencing shRNA plasmids- benefits
Guaranteed >70% gene knockdown efficiency
Control Off-target effects by multiple shRNA plasmids
Offer 4 shRNA plasmid designs for each gene
Experimentally validated shRNA design algorithm

Track/ Enrich or Select
Use Neomycin, Puromycin and Hygromycin markers to build stable cell lines and
study long term effects of gene suppression.
Use GFP Marker to track and enrich transfected cells and study short term effects of
gene suppression.
Convenient and Cost-Effective
Use standard plasmid-based and lipid-mediated transfection methods
Plasmids provide a renewable source of RNA Interference.

Accepted by thousands of scientists in various research fields
- 14 -


SureSilencing shRNA plasmids – search portal
Genome-wide collection of human, mouse and rat genes.
http://sabiosciences.com/shRNA.php

Search by Gene
Search by pathway
or disease

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SureSilencing shRNA plasmids – search portal
Search by pathway or diseases

- 16 -


SureSilencing shRNA plasmids - contents
Kit Contents:
•
•
•

4 SureSilencing shRNA Plasmids - transformation grade
One Negative Control - a scrambled artificial sequence
Sequences are provided

Additional Material Required:
•

Transformation: Competent E. coli cells & other reagents for transformation (LB,
ampicillin, plates)

•
•
•

Plasmid Purification: Plasmid purification kit, such as EndoFree Plasmid Maxi Kit
(QIAGEN Cat# 12362) and QIAfilter Plasmid Midi Kit (QIAGEN Cat# 12243)
Transfection: Lipid-mediated transfection reagent (Attractene QIAGEN Cat#301004, or
others) or electroporator; Antibiotics: Hygromycin, G418 (for Neomycin), or Puromycin
Real-time PCR Verification of knockdown:
• cDNA synthesis kit (Cat# 330401)
• RT2 SYBR Green Master Mix (Cat# 330500)
• RT2 Primer Assays – target gene of interest and a housekeeping gene

- 17 -


Workflow for shRNA plasmids knockdown
3 Steps
Start with shRNA Plasmids
GFP enrich < 1 day

1
Select with antibiotic ~1-2 week

3
2

24 or 48hr Incubation

- 18 -


Workflow for shRNA plasmids knockdown
Assay gene knockdown effect
mRNA level
• Real time qPCR, Northern blot, end-point PCR
Protein level
• SDS-PAGE and Western Verification
• Other Biochemical Assays
• Timing is critical (when do I look, when do I do experiment)

RT2 PCR Validation of gene knockdown at mRNA level
• cDNA synthesis kit, RT2 SYBR Green Master Mix
• Primers for target gene and housekeeping (control) gene such as ACTB and GAPD
• Template from transfected cells with target gene shRNA and negative control shRNA
• Perform PCR reaction
• Data Analysis

- 19 -


Real-Time PCR Validation of shRNA Knockdown

Housekeeping Gene
Control shRNA

Target shRNA

Target Gene
Control shRNA

Target shRNA

Target gene expression decreases by 2 threshold cycles, indicating >70% knockdown;
Housekeeping gene expression is not altered upon transfection with the target gene
shRNA plasmid relative to the control shRNA.

- 20 -


shRNA experiment optimization

Key points to ensure successful RNAi

Controls
Enrichment
Selection
Validation

- 21 -


Optimization #1 - controls
Untreated cells:

Use normal cells in a normal culture condition as a pure

background

Mock control:

For transfection reagent (transient experiment only). Cells treated
with transfection reagent only without any shRNA plasmid DNA. Help to identify any
effect directly from the transfection reagent

Non-targeting shRNA control:

Use the same shRNA expression vector that
will activate RISC and the RNAi pathway, but does not target any human, mouse or rat
genes. This allows for examination of the effects of shRNA transfection and RNAi
activation on gene expression. Cells transfected with the non-target shRNA vector will
also provide a useful reference for interpretation of knockdown.

This negative control is provided with each shRNA plasmid set

- 22 -


Optimization #2 - enrichment
FACS-based enrichment for GFP-expressing cells
Transient transfection may have lower efficiency in some cell lines;
Unsorted cells may exhibit lower knockdown due to a large population of untransfected
cells;
Sorting will remove the untransfected cells and enrich the population, thus providing a
true measurement of knockdown;
Peak Ex. of the GFP at 505nm, with a shoulder at 480nm; Em. occurs at 515nm

Percent
Knockdown

Pre-Sorted
Population (%)
Knockdown

Sorted
Population(%)
Knockdown

37

71.8 (69.7, 73.8)

52

70.8 (68.4, 73.0)

PRKCA
Protein Kinase C
alpha

TP53
Tumor protein
p53

- 23 -


Optimization #3 - selection
Selection using entire pool

Antibiotic selection strategy
Before transfection, determine “effective concentration” of
antibiotics
-depends on cell line, growth rate, state of confluence
After transfection, re-plate cells at a low cell density (≤10%) and
grow cells in medium containing the effective concentration of
antibiotic

Potential challenge when using pooled population
Initial stably antibiotic selected whole pool population appears to
fail, due to:

Individual Stably Selected Clones

-Random integration sites affect shRNA expression and knockdown
efficiency
-Average knockdown efficiency of all integration sites is seen, some better
than others

Strategy: Clone stably transfected cells with two best designs,
then select by limiting dilution
- Leading a high success rate: 2 out of 5 tested now successful

- 24 -


Optimization #4 - validation
To ensure >70% knockdown efficiency:
Transfection Efficiency (TE): >=80%
Biological Sample Consistency: 3

Validation – qPCR at the mRNA level
•
•
•

PCR Reproducibility
PCR Amplification Efficiency
Site-specific primer may be
necessary for some genes

100
Knocking down efficiency (% )

•
•

90
80
70
60

default primer

50

site specific primer

40
30
20
10
0
A

B

C

D

E

Designs

Validation – at protein level
At protein level, knockdown is not always immediately apparent
• Need to optimize timing
• Protein level measurement – Western blot, enzyme activity assay, reporter
assay, etc.
- 25 -


shRNA Case Studies

Application Examples – Case Studies

- 26 -


shRNA Case Study 1
Validation of STAT3 RNAi Design in-house
Target GOI (Gene of interest): STAT3 (Signal transducer and activator of transcription 3)

Assay method: Real-time RT-PCR
Cell model: 293H cells
Control HKG (Housekeeping gene): ACTB (Beta actin)

- 27 -


shRNA Case Study #1– Experimental Workflow
Validation of STAT3 RNAi Design

Transfection
Grade shRNAs

Cell Culture:
293H cells were cultured in D-MEM with 10% FBS
and 1x non-essential amino acids for no more than
15 passages.

3 x 4 GOI
+ 3 x 1NC = 15

shRN
A1

shRN
A2

shRN
A3

shRNA Delivery:
Mix 4 STAT3 shRNA plasmids (GFP) (0.8mg)
with 3mL Lipofectamine 2000 reagent in a 24well plate; Change culture media after 24hr
transfection.

shRN
A4

Check transfection efficiency by GFP expression
using fluorescence microscopy
Isolate total RNA:
Isolated total RNA after 48 hrs

Isolate RNA

- 28 -


shRNA Case Study #1– Experimental Workflow
Validation of STAT3 RNAi Design
Total RNA->cDNA
Reverse Transcription:
Synthesized cDNA from total RNA (15 samples)

15 samples

Primer set and Master Mix cocktail:
2 cocktails: GOI and HKG
Cocktail = cDNA + Master mix + H2O

Set up & perform real-time PCR:
3 technical replicates
GOI: 15 x 3 =45
HKG: 15 x 3 =45
Analyze data:
Free data analysis template performs all the calculation & generate report
http://www.sabiosciences.com/rnaidataanalysis.php
- 29 -

Analyze data & report


shRNA Case Study #1– Data Analysis & Report
DATE
11/3/2011
Transfection
STAT3 PCR 1
STAT3 PCR 2
STAT3 PCR 3
Average Ct
SD Ct
QC 1
ACTB PCR 1
ACTB PCR 2
ACTB PCR 3
Average Ct
SD Ct
QC 2
∆Ct
SD ∆Ct

STAT3-1

STAT3-2

Real-Time PCR Result for STAT3 shRNA
STAT3-3
STAT3-4

2
24.11
24.30
24.28

3
24.08
24.29
24.19

1
24.44
24.56
24.57

2
24.18
24.31
24.32

3
24.18
24.32
24.28

1
31.54
31.58
31.92

2
22.67
22.78
22.82

3
23.09
23.33
23.28

1
24.31
24.46
24.47

2
24.51
24.55
24.64

3
24.26
24.35
24.29

1
28.96
28.83
28.87

2
23.03
22.94
23.00

3
22.90
22.82
22.83

24.04

24.23

24.19

24.52

24.27

24.26

31.68

22.76

23.23

24.41

24.57

24.30

28.89

22.99

22.85

0.05

0.10

0.11

0.07

0.08

0.07

0.21

0.08

0.13

0.09

0.07

0.05

0.07

0.05

0.04

OK
17.65
17.83
17.72

OK
17.74
17.96
17.81

OK
17.62
17.70
17.66

OK
17.79
17.86
17.77

OK
17.82
17.87
17.75

OK
17.81
17.74
17.83

OK
26.38
26.39
26.45

OK
18.03
17.93
18.00

OK
18.46
18.50
18.32

OK
17.85
17.86
17.69

OK
17.87
17.94
17.84

OK
17.68
17.81
17.63

OK
25.05
24.90
24.97

OK
19.18
19.10
19.15

OK
19.15
19.12
19.16

17.73

17.84

17.66

17.81

17.81

17.79

26.41

17.99

18.43

17.80

17.88

17.71

24.97

19.14

19.14

GOI- STAT3

HKG - ACTB
0.09

OK

0.11
OK

0.04
OK

0.05
OK

0.06
OK

0.05

0.04

OK

OK

0.09
OK

0.10
OK

0.05
OK

0.09
OK

0.08
OK

0.04
OK

0.02
OK

6.39

6.53

6.72

6.46

6.47

5.27

4.77

4.81

6.61

6.68

6.59

3.91

3.85

3.71

0.10

0.15

0.11

0.09

0.10

0.09

0.21

0.09

0.16

0.13

0.08

0.10

0.10

0.06

0.05

6.41

6.55

SD ∆Ct BIO

0.11

Overall Mean SD ∆Ct

0.13

∆∆Ct

STAT3-1
STAT3-2
STAT3-3
STAT3-4

0.05
OK

6.30

Average ∆Ct

Overall SD ∆∆Ct
QC 3
Percent of Control
Percent Knock Down
+ SD
- SD
Report:

Negative Control

1
24.00
24.09
24.02

4.95

6.63

3.82

0.15

0.28

0.05

0.11

0.16

0.30

0.08

0.11

2.59

2.72

1.13

2.81

0.18

0.19

0.32

0.14

OK
0.17
83.34
1.91
2.15

OK
0.15
84.87
1.90
2.17

OK
0.46
54.24
9.02
11.24

OK
0.14
85.72
1.30
1.43

Percent Knock Down
83.34
84.87
54.24
85.72

95 % Confidence Interval
(
(
(
(

80.88
82.39
41.33
84.09

85.48
87.00
64.31
87.18

)
)
)
)

- 30 -

Design
Successful
Successful
Mediocre
Successful


shRNA Case Study #2
Case Study 2 – Published by customers

- 31 -


shRNA Case Study #2 – Cancer Research
Study: The role of PELP1/MNAR signaling in Ovarian Tumorigenesis
PELP1/MNAR (Proline-, glutamic acid–, and leucine–rich protein-1): a NR coregulator

An example using GFP marker to monitor transfection efficiency and screen the
best design, then using selection marker for long term knockdown study.

Method:
•
•
•
•
•

Model: OVCAR3 cells expressing PELP1/MNAR-shRNA;
Initially used transient transfection assay to screen 4 shRNA plasmids with GFP, and
80-90% transfection efficiency was monitored by GFP expression after 24 hrs;
Transfected OVCAR3 cells with 5 ug negative control shRNA or 2 PELP1/MNARshRNA plasmids with Neomysin;
Selected transfected cells using G418 (1mg/ml) for long term knockdown;
Assayed the knockdown effect of PELP1/MNAR using Western blot after 72 hrs

- 32 -


shRNA Case Study 2 – Cancer Research
~100% knockdown of PELP1 expression in OVCAR3 cells

OVCAR3
OVCAR3
• PELP1 expression is ~100% blocked in OVCAR3 by Western blotting; PR, cyclin D1 were down-regulated;
• The colonies of PELP1/MNAR/shRNA decreased analyzed by soft agar colony formation assay;
• The expression of Src, AKT and MAPK were decreased by down-regulation of PELP1, by Western
analysis of total protein lysates with phospho-specific antibodies

Conclusion: PELP1/MNAR plays a critical role in the proliferation of ovarian cancer cells.
- 33 -


SureSilencing shRNA Plasmids - Benefits
Efficiency and Specificity: Guaranteed success (> 70% gene knockdown by
2 different shRNA plasmids) & Minimized off-target effects
Flexibility: 4 markers, GFP, Neomycin, Hygromycin, Puromycin, allow for
transient and long-term selections
Convenient & Cost-effective: Use standard plasmid-based and lipidmediated transfection methods. Plasmids provide a renewable shRNA
resource of RNAi
Genome Wide RNAi tool for Human, Mouse and Rat genes
Search portal: Easy to search your gene of interest.
Search by gene or by Pathway or Disease
http://www.sabiosciences.com/RNAisearch.php
http://www.Qiagen.com
- 34 -


SureSilencing shRNAs for Every Gene
- A complete system for RNAi from QIAGEN
shRNAs

Analysis

Purification

Transfection

High efficiency and low cytotoxicity for DNA transfection
Suitable for all adherent cells and sensitive cells
Ideal for co-transfection and vector-based RNAi (shRNA)

Attractene

Free of animal-derived components

Fast and high-quality total RNA in minutes
Consistent RNA yields from small amounts of starting material
RNeasy Kit

No phenol/chloroform extraction, no CsCl gradients, no LiCl or ethanol precipitation

High performance: bench validated
Complete genome coverage: human, mouse, rat, rhesus macaque, fly, etc
qPCR Primer assay
SYBR® Green-based

Convenience: Within 5-minutes, deliver guaranteed performance

- 35 -


Assessing RNA Interference Phenotypes
- Cignal Reporter Assay System

Cignal Reporter Assay System
■ Dual-luciferase & GFP format
■ Plasmid based reporter assay
■ Lentivirial based reporter assay

Dicer is required in both the
siRNA and miRNA pathways
What’s the phenotypic effect of
Dicer knock down on p53
signaling?
P53 Reporter
+ Dicer siRNA

Conclusion: The regulation of p53 signaling is tightly controlled by
microRNA and/or siRNA processing.
- 36 -


SureSilencing shRNAs for Every Gene
- A complete system from QIAGEN
available for EVERY human, mouse, and rat gene
per gene set - 4 designs and 1 control
4 Marker Selection: GFP, Neomycin, Puromycin and Hygromycin

Thank You for
Attending!

Search Portal
http://sabioscience.com/RNAi.php
www.qiagen.com
www.GeneGlobe.com

Call 888-503-3187 to order
Email: support@SABioscience.com

Wei Cao: wei.cao@qiagen.com

- 37 -


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