1. 11
Actinobacteria as a Source of Novel Natural Products:
Isolation, Molecular Characterization and Phylogenetic Analysis
Khartoum, March 28th
-April 1st
Real Time PCR
as a rapid
diagnostic tool
Dr. Mogahid M. Elhassan
SUST
2. Real-Time PCRReal-Time PCR
1.1.Why Real-time PCR? Advantages andWhy Real-time PCR? Advantages and
DisadvantagesDisadvantages
2.2.Theory of Real-time PCRTheory of Real-time PCR
3.3.Types of Real-time PCR QuantificationTypes of Real-time PCR Quantification
4.4.Choosing Housekeeping Gene forChoosing Housekeeping Gene for
NormalizationNormalization
3. Disadvantage of traditional PCRDisadvantage of traditional PCR
* Low sensitivity
* Short dynamic range
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
* Ethidium bromide staining is not very
quantitative
1. Why Real-time PCR ?
4. Advantages of real-time PCR
amplification can be monitored real-timeamplification can be monitored real-time
wider dynamic range of up to 1010-foldwider dynamic range of up to 1010-fold
no post-PCR processing of productsno post-PCR processing of products
((No gel-based analysis at the end of theNo gel-based analysis at the end of the
PCR reactionPCR reaction))
ultra-rapid cycling (30 minutes to 2ultra-rapid cycling (30 minutes to 2
hours)hours)
highly sequence-specifichighly sequence-specific
1. Why Real-time PCR ?
5. 1.Requires expensive equipments and
reagents
2.Due to its extremely high sensitivity, you
may get high deviations of the same
experiment, thus, the use of internal
control genes is a recommended (in gene
expression experiments)
Disadvantages of real-time PCRDisadvantages of real-time PCR
1. Why Real-time PCR ?
7. Q-PCRQ-PCR
Definition: Real-timeDefinition: Real-time
monitoring of the amplificationmonitoring of the amplification
reaction.reaction.
Purpose: To estimate the initialPurpose: To estimate the initial
quantity of specific templatequantity of specific template
DNA.DNA.
2- Theory of Real-time PCR ?
8. The QPCR ApproachThe QPCR Approach
ChemistryChemistry
Use fluorescent dyes and probesUse fluorescent dyes and probes
Establish a linear correlation between PCREstablish a linear correlation between PCR
product and fluorescence intensityproduct and fluorescence intensity
DetectionDetection
Fluorescence detection to monitorFluorescence detection to monitor
amplification in real timeamplification in real time
AnalysisAnalysis
Software for analysis and estimation ofSoftware for analysis and estimation of
template concentrationtemplate concentration
2- Theory of Real-time
PCR ?
9. 10 X NH4 Buffer 5.0 µl
dNTP mix (12.5 mM) each 0.8 µl
Forward primer (20 µM) 1.0 µl
Reverse primer (20 µM) 1.0 µl
MgCl2 3.0 µl
Sterile Milli-Q water 38.0 µl
Taq polymerase 0.5 µl
10 X NH4 Buffer 5.0 µl
dNTP mix (12.5 mM) each 0.8 µl
Forward primer (20 µM) 1.0 µl
Reverse primer (20 µM) 1.0 µl
MgCl2 3.0 µl
Sterile Milli-Q water 37.0 µl
Taq polymerase 0.5 µl
SybrGreen (50x) 1.0 µl
Reaction contents
11. How to measure the PCRHow to measure the PCR
productproduct
DirectlyDirectly
• Sybr greenSybr green
• Quality of primers criticalQuality of primers critical
IndirectlyIndirectly
• In addition to primers, add aIn addition to primers, add a
fluorescently labeled hybridization probefluorescently labeled hybridization probe
1111
- Theory of Real-time PCR ?
16. Sybr green is a dye which binds toSybr green is a dye which binds to
double stranded DNA but not todouble stranded DNA but not to
single-stranded DNA and issingle-stranded DNA and is
frequently used to monitor thefrequently used to monitor the
synthesis of DNA during real-timesynthesis of DNA during real-time
PCR reactions. When it is bound toPCR reactions. When it is bound to
double stranded DNA it fluorescesdouble stranded DNA it fluoresces
very brightly (much more brightlyvery brightly (much more brightly
than ethidium bromide doesthan ethidium bromide does
- Theory of Real-time PCR ?
17. SYBR Green AssaySYBR Green Assay
SYBR Green
SYBR Green
SYBR GreenSYBR GreenSYBR GreenSYBR Green
SYBR Green (high fluorescent conformation)
- Theory of Real-time PCR ?
18. The TaqMan probe principle relies on the 5The TaqMan probe principle relies on the 5
´–3´ nuclease activity of Taq polymerase´–3´ nuclease activity of Taq polymerase
to cleave a dual-labelled probe duringto cleave a dual-labelled probe during
hybridization to the complementary targethybridization to the complementary target
sequence and fluorophore-basedsequence and fluorophore-based
detection.detection.
TaqMan probes consist of a fluorophoreTaqMan probes consist of a fluorophore
(Reporter) attached to the 5’-end of the(Reporter) attached to the 5’-end of the
oligonucleotide probe and a quencher atoligonucleotide probe and a quencher at
the 3’-endthe 3’-end
- Theory of Real-time PCR ?
Q RT PCR Using TaqMan
19. The quencher molecule quenches theThe quencher molecule quenches the
fluorescence emitted by the reporterfluorescence emitted by the reporter
when excited by the cycler’s lightwhen excited by the cycler’s light
source via FRET (Fluorescencesource via FRET (Fluorescence
Resonance Energy Transfer).Resonance Energy Transfer).
As long as the reporter and theAs long as the reporter and the
quencher are in proximity, quenchingquencher are in proximity, quenching
inhibits any fluorescence signalsinhibits any fluorescence signals
- Theory of Real-time PCR ?
32. Linear ground phase:
•PCR is just began
•Fluorescence emission at each cycle has not yet risen above background
•Baseline fluorescence is calculated at this time
CT - threshold cycle:
•the first significant
increase in the amount of
PCR product correlates to
the initial amount of target
template
•CT represents the starting
copy no. in the original
template
Early exponential phase:
•PCR is just began
•The amount of fluorescence has reached a threshold where it is
significantly higher than background (usually 10 times the standard
deviation of the baseline)
PCR can be broken into 4 major phases
2. Theory of Real-time PCR2. Theory of Real-time PCR
43. 4343
IL1-b
vit
RPLP0 vit
IL1-b con
RPLP0 con
av =19.80
av =19.93
av =18.03
av =29.63
∆ Ct = 9.70
∆ Ct = -1.7
∆ Ct = target - ref
∆ Ct = target - ref
Difference = ∆Ct-∆Ct
= ∆∆Ct
= 9.70-(-1.7)
= 11.40
control
experiment
44. StandardsStandards
same copy number in all cellssame copy number in all cells
expressed in all cellsexpressed in all cells
no pseudogeneno pseudogene
no alternate splicing in target PCRno alternate splicing in target PCR
region you want to amplify.region you want to amplify.
4444
4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization
45. StandardsStandards
Commonly used standardsCommonly used standards
• Glyceraldehyde-3-phosphate dehydrogenaseGlyceraldehyde-3-phosphate dehydrogenase
mRNAmRNA
• Beta-actin mRNABeta-actin mRNA
• MHC I (major histocompatability complex I)MHC I (major histocompatability complex I)
mRNAmRNA
• Cyclophilin mRNACyclophilin mRNA
• mRNAs for certain ribosomal proteinsmRNAs for certain ribosomal proteins
E.g. RPLP0E.g. RPLP0 (ribosomal protein, large, P0; also(ribosomal protein, large, P0; also
known as 36B4, P0, L10E, RPPO, PRLP0, 60Sknown as 36B4, P0, L10E, RPPO, PRLP0, 60S
acidic ribosomal protein P0, ribosomal proteinacidic ribosomal protein P0, ribosomal protein
L10, Arbp or acidic ribosomal phosphoproteinL10, Arbp or acidic ribosomal phosphoprotein
P0)P0)
• 28S or 18S rRNA28S or 18S rRNA 4545
4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization
46. StandardsStandards
The perfect standard does notThe perfect standard does not
existexist
4646
4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization
47. Applications of Q RT PCRApplications of Q RT PCR
Gene expression (and microarrayGene expression (and microarray
validation).validation).
DNA target quantification (nuclear,DNA target quantification (nuclear,
mitochondrial, residual DNA in protein prepsmitochondrial, residual DNA in protein preps
(QC)).(QC)).
SNP detection, Allele discrimination,SNP detection, Allele discrimination,
Genotyping, HaplotypingGenotyping, Haplotyping
DNA Methylation, ApoptosisDNA Methylation, Apoptosis
Viral load assays, pathogen & GMOViral load assays, pathogen & GMO
detection.detection.
Clinical Diagnostics (Cancer, TherapyClinical Diagnostics (Cancer, Therapy
Response)Response)
Notas del editor
. . . . . . . three Different things
1. Chemistry
Use fluorescent DNA binding dyes or probes to monitor the production of PCR products by fluorescence
Establish a linear correlation between the amount of the PCR product and fluorescence intensity
2. Detection
Using fluorescence detection, amplification can be monitored in real time
3. Analysis
Fluorescence data is analyzed with software that eliminates background, normalizes values and estimates template concentration
In this presentation, we will be using Sybr green to monitor DNA synthesis. Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does, which is why we use Sybr Green rather than ethidium bromide; we also use Sybr green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of single-stranded DNA is much higher that the ratio for ethidium bromide). Other methods can also be used to detect the product during real-time PCR, but will not be discussed here. However, many of the principles discussed below apply to any real-time PCR reaction.
The TaqMan Animation is a little bit tricky with the clicks. Better to try this a few times before the first presentation.
If you think the Pac Man is not serious enough, you can easily erase the face and change the pie circle to normal circle. But I have the feeling, that the Pac Man is a good link from the “normal” world to the “biotech” world, specially TAs appreciate such examples.
Meassurement in annealing phase, nothing what a ABI 7000 can do!
Scorpions is a beacon attached to one of the PCR-primers. The yellow box shows sequence idendity. The STOP sign is a PEG moity, which blocks the polymerase.
Meassurement in annealing phase, so ABI 7000 users can’t use Scorpions!
We find that the ‘PCR base line subtracted curve fit’ option (see area inside pink box on the slide) which is the default analysis mode in the current version of the Biorad Icycler program (3.0a) Does not give such good results as the ‘PCR base line subtracted’ option. So we always use the latter.
As we saw with the theoretical curves, you should get a straight line relationship in the linear part of the PCR reaction. In this case the reaction is linear from ~20 to ~1500 arbitrary fluorescence units.
There are several methods to quantitate alterations in mRNA levels using real time PCR, let’s look at the standard curve method first.
In the following discussion the results shown will be those obtained in our laboratory using a BioRad Icycler Real-time PCR instrument. However, analysis with other instruments is similar.
Here is a real-time PCR trace for a single well on a 96-well plate, cycles are shown along the X-axis, and arbitrary fluorescence units (actually these are fold increase over background fluorescence) are shown on the Y-axis - the results are similar to our theoretical graph (see insert) - except that the transition to the plateau phase is more gradual. This expt - and everything we are going to discuss - was done with SYBR green, which has very low fluorescence in the absence of double stranded DNA and very high fluorescence in the presence of double stranded DNA.
Here is the data from our dilution curve. If you are looking at efficiencies, you want to be sure that every time you do the PCR for the same gene you have the same slope since this is a measure of efficency - in this case you can see that all the samples are reasonably close (the lines are all parallel). If there is a difference in slope of one of your samples, it implies a problem in that tube (PCR inhibitor, problems with enzyme, etc).
This shows the same data as on the previous slide but on a logarithmic scale. The even spacing of the reactions is now much more obvious. So what the software actually measures for each well is the cycle number at which the fluorescence crosses an arbitrary line called the threshold - shown in orange. This crossing point is known as the Ct value. More dilute samples will cross at later Ct values.
Similarly, you can select the wells in which you amplified the reference gene and determine the relative amounts in the experimental sample compared to the control. This will give you the bottom value in the “Northern formula” from slide 4.
Now that you have both values, you can divide the target gene value (purple) by the reference gene value (blue) and obtain the ratio of the target gene in the experimental sample relative to the control sample, corrected for the reference gene (loading control).
This method will give the fold changes in the target and reference genes - so one can calculate a fold change corrected for any variations in the reference gene. The disadvantage is that you need a good dilution curve for both standard and reference genes on every plate - which would be at least 16 extra wells (including negative controls) for us. If there is any problem with either dilution curve, the data cannot be analyzed, or a suboptimal curve has to be used. So -- we prefer to use determine efficiency accurately (on multiple days) and then take an average of multiple results and use these separately - this makes experiments simpler but we need to think a bit more about the maths of calculating the results because this time the machine doesn’t do it for you.
We find that the standard curves are highly reproducible if you use a supplier who provides a mix with stablizer(s) for SYBR green.
This method was one of the first methods to be used to calculate these type of results. However, as we shall see, it is an approximation method. It makes various assumptions, and to prove that they are valid is, in our opinion, more time consuming that doing a few extra efficiency runs for the Pfaffl method.
If one looks at the same data that we discussed before. But for the time being ignore the data from the standard ‘loading control’ gene. The difference between the control and treated samples for il1-beta is shown by the red line. If we know the efficiency for il1-beta and the cycle number, we could calculate the fold change in il-1 beta- but there would be no loading control.
An approximate correction can be made for the loading control by calculating the difference between the IL1-beta Ct values and the RPLP0 values for the control samples, and then for the vitreous treated samples (represented by the two green lines in the slide). This makes an allowance for the fact that in the above case, there is slightly more mRNA in the vitreous treated samples (since the RPLP0 comes up slightly earlier). This difference (or delta Ct value) is shown by the two green lines above. The difference between the two delta values represents the shift as will be seen in the next slide.
The difference between the two delta Ct values (delta delta Ct, represents the corrected shift of the IL1-b. Since in this example in the vitreous treated sample the IL1-b has moved to the left of the standard, it has a negative value, but in maths subtraction of a negative value is equivalent to adding that value - which makes obvious sense (I hope) if you look at the diagram - the total shift is = the two green arrows added together. If the vit il1-b had shifted but remained to the right of the reference curve, the value would then be subtracted from the large green arrow to determine the shift.
However, the perfect standard does not exist, therefore whatever you decide to use as standard or standards should be validated for your tissue - if possible you should be able to show that it does not change significantly when your cells or tissues are subjected to the experimental variables you plan to use.
