Dear all
to measure the mRNA level of a monoexonic gene, we cannot design specific primers to overlap any introns. Thus, we can follow the "two-step" RT-PCR and then compare PCR results from 1) reverse transcribed RNA and 2) non-reverse trascribed RNA. If the non-reverse transcribed RNA has amplification, it means that has a genomic DNA contamination.
Is there another way to do it? quicker?
Make primers for the intron only and the exon only.
Use the expression of the intron to adjust for DNA contamination.
Asif
You can use DNase I to wipe off the genomic DNA contamination. Then inactive the DNase with EDTA at 80℃ for 2min. Non-Dnase I treated RNA is control sample.
Hi,
I agree with Peng. Although the DNAse treatment is rarely 100% efficient, this should be surely done. And I guess you probably did it.
Once, when we wanted to do similar comparison of RT-PCR with and without reverse transcription in one-step RT-PCR, we just added the transcriptase-polymerase mix into "no-RT tube" after the step of RT, just before PCR. Maybe this could be a simplier way for you.
All the best, Lucie
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