Are you new to using SYBR Green Assays for qPCR or having trouble getting accurate results? Today, let’s discuss how you can design and optimize qPCR using SYBR Green assays as the detection method.
First, beware of reverse transcription (RT) bias when converting RNA to cDNA. Nearly all RT enzymes have the potential to introduce RT bias. When this happens the amount of cDNA won’t align with the amount in RNA samples. Learn More about RT Methods in this video: What is the Right RT Method for Your qPCR Experiment
You can test for RT bias by reverse transcribing two-fold dilutions of a known amount of RNA. Then run a qPCR standard curve for each assay and endogenous control. The standard cure should be linear with a target slope of -3.323.”
Once the cDNA is generated make sure to use the right primers for the qPCR.
You will need to use some bioinformatics to design your primers, such as a tool like SNPMasker.
In general, primers should be 20 nucleotides in length with a GC content in the 30-70% range. The last 5 nucleotides at the 3’ end should include no more than two G or C bases to avoid specificity issues. Finally, amplicons should be short — generally between 50 – 150 base pairs.
The next step is primer validation. The objective is to find the right concentration of forward and reverse primers that will yield the most robust assay without non-specific amplification or primer-dimers.
This is accomplished by running multiple qPCRs with 3 different concentrations of forward and reverse primers in a matrix format. The appropriate range of primer concentration is determined by the master mix.
For instance, Applied Biosystems PowerUp SYBR Green Master Mix works best with primer concentrations in the range of 300 – 800 nM.
Getting back to our experiments to optimize primer concentrations, the next step is to evaluate the Ct and run a melting curve also known as a dissociation curve for each primer concentration combination.
If dissociation curve shows primer-dimers, there are two options:
A. Start over and redesign the primers.
B. Alter cycling temperatures to remove primer-dimers.
The last step in ensuring that your primer set is going to yield usable, reproducible data is to ensure the PCR efficiency is within 90 – 110%. You can do this by simply running a standard curve with at least 5 logs of input DNA and using the software on your instrument to calculate PCR efficiency.
If this all seems too complicated, you can use pre-designed TaqMan assays instead, which removes the primer design variable and ensures the best possible primer set
Once the primers are designed, experimental analysis can begin.
Here’s a tip! For measuring relative gene expression levels of two different samples, most researchers use what’s known as the ΔΔCt (ddCt) method. This analysis generates relative changes form one state to the next, much like a disease state versus treatments
For more information about SYBR Green experiments, TaqMan assays or related reagents, please visit http://www.thermofisher.com/qpcr or http://www.thermofisher.com/sybr
And If you have more questions concerning SYBR green assays, ΔΔCt analysis or any other qPCR questions, remember to Ask TaqMan and submit your questions on our website http://www.thermofisher.com/ask
Thanks for watching!
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