genomic dna in real-time pcr - (Mar/07/2006 )
my RNA was run through qiagen rna column and then treated with Dnase I (invitrogen). however, i still got signal for using a pair of primers designed from the genomic sequence. i really dont know what to do to deal with this.
for example, the ct of beta-actin primer from mRNA sequence was 20 and the ct of beta-actin primer from genomic sequence was 32. does this really matter? could i dilute the template to minimize the effect of the genomic dna? has anyone had the similar problem?
I tried to purified RNA using Qiagen columns also with run TRizol reagent, from macrophages and from bacteria, I used the DNase Qiagen treatment onto the column and DNA still there, I run all the time PCR after DNase treatment. I used the Dnase from Promega, it can eliminate all DNA at high concentration, problem: sample is diluted, and maybe RNA is degraded? now we are going to try with DNA fre from ambion, will see.
Could you read next: Quantification of mRNA using real-time reverse transcription PCR:trends and problems, S. A. Bustin. J. of Molecular Endocrinology (2002) 29:23-39,
To eliminate the effects of genomic DNA make sure your primers are intron spanning, so they shouldn't amplify anything from genomic DNA, you can always test primers on genomic DNA to check if they do amplify it or not.
I've had good luck with both on-column DNase digestion (Quigen) and using the 'DNA free' reagents from Ambion. Particularly with the Ambion kit, you should be careful to get a sense of DNA contamination and adjust the amount of DNase and incubation time needed to clear the samples.