I am attempting to validate existence of a transcript using 40 cycle qPCR. I designed primers for a unique feature of this transcript, and also designed primers for a sequence in the transcript that is common to several transcripts of this gene, and finally I used GAPDH as a control. I already knew beforehand that this transcript has very low expression.

For the unique region, my ct values for primer 1 were between 31-34.8, and my ct values for primer2 and primer 3 were all consistently 35, and primers 4-6 gave no result at all.

For the region that is common to several transcripts of this gene, the ct values were between 30-34 for one primer, and consistently 35 for another primer.

For GAPDH, ct values were around 22.

Can I use these results to indicate that my transcript exists (or even may exist?) in the samples I tested? Is the fact that the ct values for the region unique to the transcript I am targeting and the region in common between several transcripts are quite close indicative of anything? On the graph from the qPCR, there are distinct rises respectively around 33 and 35 for the unique region amplicon.

EDIT: I was doing a bit more research, and it appeared that this study: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296690/
also had results that came up "late cp call last five cycles has higher uncertainty" and considered these results positive if they could be replicated in a duplicate - if I were to show this as well, can I use this data in a paper?

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    $\begingroup$ What have you done to validate the conditions of your reverse-transcription and real-time PCR with respect to these transcripts? Can you get more input mRNA (say, enough so that $C_t$ values for GAPDH are well under 20)? Is the final PCR product for your transcript-specific primers at least the correct size based on your predicted transcript? Note that $C_t$ values differing by 4 units represent 16-fold differences in transcript abundance. $\endgroup$
    – EdM
    Jul 18, 2015 at 0:11
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    $\begingroup$ @pMarkov you validate PCR product size by running regular old-fashioned PCR with your primer pair of interest, and run the reaction out on a gel with a ladder for reference. $\endgroup$
    – MattDMo
    Jul 18, 2015 at 1:46
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    $\begingroup$ If you can't replicate the biological experiment, all the re-analysis of old cDNA isn't going to be good enough. Reproduce the conditions that led to the hypothesized production of the novel transcript, at a scale that allows better control of PCR conditions. $\endgroup$
    – EdM
    Jul 18, 2015 at 2:01
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    $\begingroup$ No prob. Sometimes we get too caught up in all the technology in the lab and forget about the simple stuff... $\endgroup$
    – MattDMo
    Jul 18, 2015 at 2:02
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    $\begingroup$ To validate the existence of transcripts could be done by sequencing of PCR products amplified if you can deny contamination and amplification from genomic DNA. $\endgroup$
    – 243
    Jul 18, 2015 at 3:00


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