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.
