2 replies to this topic

[themoon]

Hi,
I wanted to know if genomic DNA contamination is a problem even if the primer itself designed on the exon-exon boundary? Since the gDNA wouldn't amplify at all if they were designed that way then should I forget about doing DNase treatment?

Thanks heaps

[ivanbio]

If the assay was designed to span an exon-exon junction, then in theory you do not need to worry about DNA contamination. Having said that, I would still try to purify the RNA in the best way possible, including DNAse treatment. One reason for this is that when you quantify your RNA you cannot tell RNA from DNA, so any DNA contamination will show up as if having more RNA (total nucleic acids). Plus, the presence of DNA (and other contaminants like protein) will more often than not affect your assay in a negative way.

My two cents

[pcrman]

No matter how your primers are designed, efforts should be made to avoid or eliminate DNA contamination. If you are going to use the primers in qPCR, primers' spanning exon-intron junction won't help because the machine cannot tell you the amplification is from cDNA or DNA.

If you do regular RT-PCR using such primers, DNA contamination can be found by the existence of a high sized band from the DNA. Despite that, If your purpose is to compare gene expression by judging the intensity of the bands and if the DNA contamination is serious, the amplification from the DNA may interfere with cDNA amplification and makes your interpretation of gene expression level inaccurate.