Hi everyone,
I am analysing the gene expression from different Drosphila genes by RT-qPCR. I am having problems with one of the gene, OR24a, because I see amplification in the "No RT" sample, that did not happen with the other genes. This gene is particularly small.
The primer pair that I used, has a 21 bp size, was designed with span exon-exon, 50% GC and has a melting temperature of 62ºC. The amplicon has around 73 bp.
I used the Qiagen QuantiTect Reverse Transcription Kit, that includes a treatment with DNAse. I also tried a previous treatment of RNA samples with RNeasy® MinElute®
Cleanup, but no sucess. For the RT-qPCR I used the SYBR Green from applied biosystems: enzyme ativation at 95ºC 10 min; Denature 95ºC 15 sec; Anneal/Extend Temp. 60°C 60 sec. (with melting curve, 40 cycles). I also tried the sSoFast Evagreen from Biorad: enzyme ativation at 95ºC 30 sec; Denature 95ºC 5 sec; Anneal/Extend Temp. 60°C 5 sec. (with melting curve, 40 cycles).
I suspect of genomic DNA contamination, but I obtained exactly the same melting temperature in the melting curve with the positive sample and "No RT sample".
Any suggestion about what is happening and strategies to overcome this problem?
Thanks in advance,
Have you considered aerosol amplicon contamination for the problem gene? I have seen it happen before.
Hello. Thank you for your pertinent sugestion. However, I did not see amplification in the "No RT" sample for the housekeeping gene, that I ran in parallel with Or24a gene.
You don't need to get a contamination of the reference gene. Perhaps there are Or24a amplicons floating around the lab in the air. I have seen runx1-runx1t1 gene fusion show up in a diagnostic assay for several replicates of NTC even though there were other gene fusion assays in the same test being multiplexed. If someone has used those primers before, definitely be wary of that possibility. The other alternative to aerosolized contamination is labware/consumable contamination. Try with a fresh set of reagents and labware if you can, if the NTC is OK, then you know it was not in the air but something to with a mix/pipet etc. I really hope that it is not the air.
BTW, are you using exon junction primers so that introns are not amplifiable? That would suggest the odds of gDNA contamination to be low if your primers bind to exon-exon junctions.
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