In my experiment, I want to test mRNA level by qPCR. First I isolated total RNA from mice cerebrum tissue and then did both 1-step qPCR and 2-step qPCR. I can clearly see there is a peak in "no-reverse transcriptase" group, in which there is supposed no peak.
I think the reason is there is gDNA contamination in RNA. Even though I've tried many ways, such as 1. double DNase concentration and DNase treatment time, or 2. DNase treatment during or after or during/after RNA purification, peak is still in "no-reverse transcriptase" group.
I also tried extracted RNA by Ambion Kit and Trizol. It seemed both did not work.
Could anybody give me some advices? Thanks.
Since I have never heard of this before, could you please share your protocol for the "no-reverse transcriptase" group? Are you using this as a control?
Regarding the gDNA contamination: Did you check if your primers are also specifically amplifying any gDNA sequence? And where did you design your primers? Inside of an exon or exon-junction-spanning?
Two thoughts:
First, it could be contamination from genomic DNA. Do your primers span exon junction?
Second, it could be primer dimers or another artifact from the PCR. Try running out your products on an agarose gel to see what size products you find in your reactions.
Good luck!
I would say as the others :
-either your DNase digestion did not remove all gDNA, and if your PCR primers don't span an intron and/or an exon junction, then you can generate the same amplicon (same length) from cDNA and from gDNA (in No-RT negative controls) ;
-or all gDNA was destroyed and then the signal in no-RT controls can come from two sources : (1) primer dimers (so, compare de fusion peak to the one from PCR Blank) ; (2) cross-contamination between PCR wells during pipetting (aerosols of cDNA came into no-RT wells).
Overall, the way of understanding the origin of signals in qPCR negative wells is (1) using SYBRGreen reporting,(2) comparing the fusion peaks from PCR Blank (water), no-RT (gDNA from RNA) and cDNA, and (3) comparing the electrophoresis patterns from these three kinds of products.
At last, even if your negatiive controls give a signal, the main point for qualifying or not your gene expression results, is the Cq i.e. the relative quantity of artifactual vs positive signal : if your No-RT and Blank occur at Cq 35 and your cDNAs at Cq 20 to 25, then no risk at all that these gDNA and primer dimers artefacts could impact your gene expression measurements. However, low-expression transcripts could generate Cq closer to the artifactual Cq, then you should choose other primers and optimize your PCR conditions. For these reasons, I always run replicates of all negatives controls together with the samples in any gene expression qPCR plate.
Best regards,
Claudine.
Hi Claudine,
Thank you for your kindly response.
-The primers does not span intron, because there is only 1 exon in DNA. So I have to design primers during this exon.
-For primer dimer, I don’t think the peak will be. Because the peak in “no-RT” group and “RT” group have the same melting point. They are almost the same. For cross contamination, I set a group in which water is used as template, and no amplification curve and dissociation curve exist. So the cross contamination can be excluded.
As you said, the No-RT Cq is over 30, the gDNA is supposed to be no harmful for detecting other gene expression. So I moved forward.
Thank you so much for your advise.
Best,
Yao.
Hi Katie,
No, primers don’t span intron because there is no intron at all... Only 1 exon.
The PCR product from “no-RT” group has the same size band with that from “RT” group in agarose gel, so it must be gDNA contamination, however, the Cq is over 30, so I just moved forward and I neglected it.
Thank you very much for your response.
Best,
Yao.
Hi Ariane,
The primers are specific. I designed them by NCBI Primer blast, and because the extension time is short, the other longer product will not be amplified. The primers are inside an exon because there is only 1 exon, I have no choice when I designed the primers.
Thank you for your response.
Best,
Yao
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