Hi to everyone! I have been having an issue with my retrotranscription negative controls (I have 2 samples and it happens in both). I am using a GAPDH primer (F & R - 500 nM) for amplification which anneals to the RNA transcript and gives a 106 bp long product. I revised and the primer also anneals to the gene and it theoretically gives a 235 bp long product. The problem is that I ran a gel with my samples (A and B, Fig. 1) and their respective negative RT controls (1 and 2, Fig. 1), which contain RNA but not the retrotranscriptase, and if there is any amplification, it is supossed to be because of contaminating DNA, but the product should be 235 bp long, not 106 bp, because there is, theoretically, no cDNA synthesized.
I also ran another gel with amplicons formed directly by a DNA sample with a rep (1 and 2, Fig. 2), so I thought the product found should have been 235 bp long (I gave it 15 sec of extension using a Hot Start pol), but I did not found the expected 235 bp long product, instead I found the 106 bp long product and others (I used gDNA). So I am really confused. Hope anyone could help me. Thanks!
It sounds like pcr amplimer contamination of your reagents or system.Do you get an amplimer if you set up a pcr using water as the template but no cDNA or genomic dna?
Hi Paul Rutland, thanks for your answer. Actually in Fig. 1, the next two lanes on the right of the Negative RT controls are the RT blank (no RNA) and PCR blank (no cDNA). So as you can see, there is no amplification, only the primers are visible on the bottom of the gel very diffused. Additionally, the pPCR shows no amplification for both blanks at 36 cycles. So the issue is my own sample.
I also did a qPCR directly with my RNA samples and it amplified the same product size (106 bp) but very low copies. I do not really know what am I failing at analyzing.
It seems to me Antonella Sialer that figure 1 shows amplification at 106 in samples a and B (which should be negative for 106 amplification) but also controls 1 and 2 on the RHS of the ladder are also both amplifying 106 when they should be blank and the only reason that I can see for this is that some pcr product of 106bp has got back into your reagents and the primers are re amplifying this contamination. It is odd that qPCR is not showing the same problem but if the contamination is in a reagent that is used in the ordinary PCR but is not used in the qPCR then that result is possible
Hi Paul Rutland , thanks for your answer. I kind of solved it out, and there was no contamination. The issue here is that the primer (which I did not design) binds to another 2 regions in other chromosomes (I had to run a PCR in silico to find it out) which give a 106 bp long products each, so that is why mi primer amplifies in my -RTC, given the traces of contaminating gDNA after purification. I imagine that the other products I found with gDNA directly are inespecific products becase of the 30 seconds I gave it to extend (I am using a Hot start Taq which is much faster).
well done Antonella Sialer I am delighted that you have solved the problem
best wishes Paul
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