I'm trying to determine a strategy to study the expression of certain parasites' biomarkers in different conditions.
Firstly, I do not know if the best approach would be to start with common PCR or immediately design the Taqman probes for RT-PCR.
How important is it to study the viability of the chosen gene region/selected primers by common PCR, before RT-PCR methodology?
For example, in common PCR, if there is amplification of the cDNA internal control, and no amplification of the interest gene, does that mean my designed primers are not viable, or can it mean that there is a diminished expression of that gene/protein in my sample?
I hope you understand the questions and will be able to help me, thank you.
Dear Andreia, maybe You should do melting in the last step of Real-Time PCR, which informed us about specificity of the primers. Moreover, I think that in Your cDNA, the targed gene may not be expressed. EW
Edyta thank you for your comment. I was afraid that I needed to test my primers by common PCR first, and only then to select the better ones to create the probes for RT-PCR. Indeed the melting curve, might give me the necessary information about specificity regardless of sub/overexpression. On the other hand it might become a bit expensive to buy a library of probes for RT-PCR to choose a small set of them....
Hi
After your primer designing, try to check your primer with positive control (The sample which is expressing your target gene). Then you can sort out the issue, the problem is with sample or your primer design.
Design your PCR primer of about 300-400bp. Once you successfully amplified your targeted gene in the positive control, conform the product with sequencing. Once you confirmed, You can design realtime PCR primer within the 300-400bp product. So your RT-PCR should work well.
If have more sample you can go for SybrGreen qRT-PCR, it is cheaper than Taqman.
Good luck
Anand
Hi Andreia, first of all, you should check the target gene expressed in your sample probably by literaturing. Low expression level of the target gene will not give you positive results in most of cases. Thus, you may need a positive sample, which you are sure that the target gene is definitely highly expressed. Use this positive control to test your designed primers. A single band after gel electrophoresis indicates your primers work well. Typically, according to my experience, 100-150 bp length of PCR products give great efficiency, which is very important for your calculation. If you are doing hundreds of samples but several genes, Taqman may overmatch SyberGreen. Otherwise, try to go with SyberGreen, cause it is much cheaper and the results won't be bad.
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