Hello.
I would be thankful if anyone could answer my question.
We are Analyzing the expression of a certain gene in our research.
We have extracted all the mRNAs in the cell and now I was wondering if we should Perform a PCR before doing real-time PCR? ( And Not for the purpose of quality check and control, But for the purpose of extra-Amplification)
wouldn't doing PCR on Our cDNA targets before real-time PCR result in a false increased Data about our gene amount?
You are correct. You should always perform PCR before new rt-PCR, but its for the purpose of quality check, control, checking for possible contamination, and if you want the product sequenced, not for extra-amplification. If the problem is the quantity of the gene and late amplification, you can increase the cDNA concentration during rt-PCR.
It is key for one to carry out PCR first before rt-PCr, in the sense that, the rt-PCR helps you know more about a detailed expression of the gene. It could be that bands will show from the PCR but with the rt-PCR, it helps you know more, maybe in percentages or (any other quantitative approach) which tissue showed more expression of the gene and also for quality check purposes.
the purpose of reverse transcription PCR is to covert your mRNA into DNA. I would do this first. However, if you are not going to use the sample for qPCR afterwards you could do the reverse transcription as a PCR like step and amplify your gene of interest at the same time. I think it is much more common to just do regular rt-PCR and then do any other downstream analysis (qPCR, PCR, sequencing etc) on the cDNA.
Hello,
I always suggest to run a PCR before RT-PCR to the ims of:
be sure about expressed genes, size of genes in all genotypes, see the quality of genes and most important think is to see the primers quality.
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