Hey, everyone,
My PCR results with two bright binds puzzeled me these days. My primers are designed by Primer-Blast of NCBI and compared to mRNA-Ref database, the expected band is 200bp. However, I found another bright band between 400bp-500bp. I tried to BLAST my primers with genomic DNA database, and found maybe the results comes from genomic DNA contamination, which suggested about 457bp. But, I can not figure out that my primers are span exon designed, will it be possible that my RNA contaminated wih genomic DNA ? In my RT-PCR assay, we used oligo dT to change RNA to cDNA, and use cDNA as template to PCR. Will the genomic DNA still exist after RT-PCR process? So, can my primers be used in real-time PCR assay ?
Hope someone answer my question, much thanks.
Hi,
did you include a DNAse step in your RNA extraction? I usually do this and I never had a problem with gDNA contamination. You should be able to see the DNA on a gel when you check the quality of your RNA extraction, as a very large-size band that goes away after DNAse treatment.
Also, is there a possibility that mRNA or ncRNA versions of your ranscript exist with retained introns? Might be an option.
How do you extract your RNA? If you use Trizol/Chloroform, try doing another chloroform extraction.
I use DNAse treatment during RNA extration or prior to cDNA synthesis to avoid genomic DNA contamination. I use rDNAse I form Ambion. Do not forget to inhibit DNAse before cDNA synthesis to not degrade your cDNA.
Hi, i think your problem related with suitable controls, Kamil Krawczyński give you a good options....good luck
use DNAse before RT-PCR to digest genomic DNA.
or this problem may be due to alternative RNA splicing
I agree with the hypotesis of genomic contamination.
Try to understand if your primer positions on genomic sequence comprise an intron of 250 bp long... and the mystery will be unveiled... anyway, the suggested controls are mandatory.
answers: of course, genomic DNA easily "survives" to RT; your primers are very little useful for real time PCR (your melting curves could be very populated).
you need better primers: an excellent primer design can even make tolerable some inevitable DNA contamination...
Two things can be done to avoid genomic DNA amplification in the PCR results,
1. Avoiding DNA contamination by doing in column DNase digestion of DNA or treating the RNA with DNase and cleanup if RNA is isolated by Trizol method. However the latter method yields in low RNA concentration.
2. Design primers in the exon-exon junctions using primer 3 tool. Put a hyphen in between exon-exon junctions in the sequence before pasting the sequence to primer 3 tool. Make sure that there are no complimentary regions for primer dimer and hairpin formation.
All the best.
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