1. A short overview on the real-time quantitative PCR technique
Real-time reverse transcription (RT) polymerase chain reaction (PCR) is the most sensitive and reliable method for the detection and quantitation of nucleic acids levels.
The RealTime PCR Instruments allow real time detection of PCR products as they accumulate during PCR cycles.
The figure below shows a representative amplification plot and defines the terms used in the quantitation analysis. An amplification plot is the plot of fluorescence signal versus cycle number. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline. The parameter Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
So the higher the initial amount of the sample, the sooner accumulated product is detected in the PCR process as a significant increase in fluorescence, and the lower the Ct value. Ct values are very reproducible in replicates because the threshold is picked to be in the exponential phase of the PCR, where there is a linear relation between log of the change in fluorescence and cycle number and the reaction components are not limiting.
2. Chemistry
a. 5’ nuclease assay (TaqMan ):
It involves the 5´ -> 3´ nuclease activity of Taq DNA polymerase. A probe carrying two dyes (one of them is a fluorescent reporter dye and the other is a quencher dye) hybridizes to the amplicon during the PCR reaction. The two fluorescent dyes interact whenever the probe is intact, causing the quencher dye to quench the reporter dye. During the amplification, the Taq DNA polymerase cleaves the 5´ end of the probe, releasing the quencher dye and results in an increase in fluorescence.
The change in reporter dye fluorescence is quantitative for the amount of PCR product produced
The Service uses a nonfluorescent quencher dye (NFQ), which raises the signal –to –noise ratio, linked to the minor groove binder (MGB), which allows to make shorter and more specific probes with the same Tm. The reporter dyes are usually 6-FAM for the target gene probe and VIC for the housekeeping probe.
b. SYBR Green I dye
The SYBR Green I is a double-stranded DNA (dsDNA) binding dye which is incorporated in the amplicon during the PCR. When SYBR Green I dye binds the Minor Groove of dsDNA, the intensity of fluorescence emission increases.
All the PCR products (including the non-specific ones) are detected in this way, but no probes are required.
3. Working scheme
The scheme below represents all the steps involved in the samples processing
Step1 and Step2: See Sample preparation in the page after logged in
Step 3: : The User must control if the genes they are looking for were already tested or not by the Service, searching in the “Is your gene already tested?” tool orthe “Target Genes Table". Then they have to contact the Service to choose the best strategy of working: the Service will provide to order the assay (see Assay selection in the page after logged in)
Step 4: Before starting the whole experiment, the cDNA quality is tested, running only the 18S gene on all the samples and their RT- controls, checking their Ct value. In this way we can decide:
- if there is any significant genomic contamination
- the best concentration to work with.
If the samples are good, the cDNAs are divided into aliquots of the correct concentration before freezing them at -20°C
Step 5: For this kind of chemistry it necessary to verify the amplification efficiency of the target and the housekeeping gene using standards curves. Moreover it is necessary to identify the primers concentrations that provide optimal assay performance. This enables to compensate for non-specific primer binding, which can reduce the amount of primer available to bind at its specific site, by using the primer optimisation matrix shown in the following table:
|
|
Forward Primer (nM)
|
Reverse Primer (nM)
|
|
50
|
300
|
900
|
50
|
50/50
|
300/50
|
900/50
|
300
|
50/300
|
300/300
|
900/300
|
900
|
50/900
|
300/900
|
900/900
For SybrGreen assay, optimal performance is achieved by selecting the minimum primer concentrations that provide the lowest CT and highest deltaRn for a fixed amount of target template, without non-specific amplification.
The primer optimisation matrix should be performed with at least two different amount of cDNA (to exclude concentration depending phenomena) and run with NTCs (no template controls) to check for primer dimer non-specific product.
Dissociation curves is extremely useful when selecting optimal primer concentrations for a SYBR® Green I, especially to check for the presence of primer dimer product. This is demonstrated in
Figure 1. Amplification data using SYBR® Green I assay chemistry
(a) amplification plot (linear view) demonstrating suspected non-specific amplification in NTC (no template control) wells.
(b) Dissociation curve analysis confirming product in NTC wells has a different melting temperature from specific product. Dissociation curve analysis is performed after a completed PCR. Data is obtained by slowly ramping the temperature of reaction solutions from 60 to 95 °C while continuously collecting fluorescence data. The increase in temperature causes PCR products to undergo denaturation, a process accompanied by a decrease in fluorescence for solutions containing SYBR® Green I dye
The strong amplification of the NTC wells shown in Figure 1(a) indicates that significant non-specific amplification is occurring. This is confirmed by the dissociation curve data shown in Figure 1(b), which shows that the melting temperature of the product generated in the absence of template is lower than the melting temperature of the specific product generated with template. This is typical of primer-dimer formation and indicates that lower primer concentrations should provide more optimal results with a larger linear dynamic range.
Step 6: We work in triplicate and include NTC (No Template Control) during the amplification step.
All the assays run using the same universal thermal cycling parameters, for both TaqMan® or SYBR® Green I chemistry, as follow:
Hold 2 min 50 °C( optimal AmpErase® UNG activity )
Hold 10 min 95 °C( AmpliTaq Gold® DNA Polymeraseactivation and UNG inactivation)
For 40 cycles: 15 sec 95 °C 1 min 60 °C
Step 7: The Instruments set-up and the amplifications are performed following the ABI 7900HT sequence detection system and the ABI PRISM 7700 sequence detector Analyser User’s manual and analysed with the SDS 2.1.1 software.
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