Hello,
Is that a must that I digest my RNA sample with DNase during purification? I am using Qiagen RNeasy protect mini kit. Further, I will be using SYBR green qPCR kit. My RNA target does not seem to be of low abundance. Would the reviewers mind if I don't digest my RNA sample with DNase?
Best,
Nanda
Hi Nanda,
You are leaving out a major clarification (Dr. Nadine Gund would agree here).
If you are working with eucaryotic DNA and you are absolutely certain that you have designed your primers to a genomic exon:intron junction (with 100% confidence), you should be fine without using the safety net of a DNase treatment here, in theory.
If you are working with procaryotic RNA, you have an absolute requirement of DNase treatment of RNA since procaryotes lack introns.
What kind of RNA are dealing with? Reviewers will like to know that - up front.
One must always assume that there will be gDNA contamination with every RNA isolation method. Then, it is up to you to decide how much fidelity your primers actually have for your target based on the concerns stated.
Hi Nanda,
You are leaving out a major clarification (Dr. Nadine Gund would agree here).
If you are working with eucaryotic DNA and you are absolutely certain that you have designed your primers to a genomic exon:intron junction (with 100% confidence), you should be fine without using the safety net of a DNase treatment here, in theory.
If you are working with procaryotic RNA, you have an absolute requirement of DNase treatment of RNA since procaryotes lack introns.
What kind of RNA are dealing with? Reviewers will like to know that - up front.
One must always assume that there will be gDNA contamination with every RNA isolation method. Then, it is up to you to decide how much fidelity your primers actually have for your target based on the concerns stated.
Hello,
Thanks. I am working with a eukaryotic gene and my primers fall in the exon region. I was checking the protocol from Qiagen and it said generally DNase digestion is not required. And I am hesitant to do the digestion as I don't want my samples to degrade. Would there be any other way to work around the problem? I appreciate your answer!
Best,
Nanda
Qiagen is worried about residual RNAse in their DNase reagent (a common worry).
Turbo DNase is 350X more catalytically active than wild type DNase I - that may be another option for you.
Or, if you stick with the Qiagen DNAse, don't prolong the digestion time.
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