Problem with DNase digestion of RNA - (Jul/20/2006 )
Hello
I use to isolate my RNAs with Trizol and then to digest with DNase from Promega, before performing reverse transcription and real-time PCR.
But sometimes unfortunately I lose samples in concentration and in purity as well when I measure absorption after digestion (using a Nanodrop). So I wonder if it worth it... I mean, I designed my primers and probes for real time so that they span intron-exon junctions to avoid genomic amplification. Is it not sufficient? Do I really need to digest the RNAs anyway? I know it is always better to do the more than the less, to get the most reliable results at the end. But if I lose in quality... what's the best solution??
I 've thought I could try to run one real time PCR with a raw RNA sample and a digested one to see if there is any difference in amplification. It could be an easy control as I don't have any genomic DNA to use as negative control.
Could someone help me, even in these hot sommer times (in Europe at least)?
thanks in advance
I use to isolate my RNAs with Trizol and then to digest with DNase from Promega, before performing reverse transcription and real-time PCR.
But sometimes unfortunately I lose samples in concentration and in purity as well when I measure absorption after digestion (using a Nanodrop). So I wonder if it worth it... I mean, I designed my primers and probes for real time so that they span intron-exon junctions to avoid genomic amplification. Is it not sufficient? Do I really need to digest the RNAs anyway? I know it is always better to do the more than the less, to get the most reliable results at the end. But if I lose in quality... what's the best solution??
I 've thought I could try to run one real time PCR with a raw RNA sample and a digested one to see if there is any difference in amplification. It could be an easy control as I don't have any genomic DNA to use as negative control.
Could someone help me, even in these hot sommer times (in Europe at least)?
thanks in advance
I don't digest with DNase and I don't have problems. Invitrogen says you should digest with DNase only if you are using primers that anneal in 1 exon, because you should have almost no contamination from DNA.
Hello
I use to isolate my RNAs with Trizol and then to digest with DNase from Promega, before performing reverse transcription and real-time PCR.
But sometimes unfortunately I lose samples in concentration and in purity as well when I measure absorption after digestion (using a Nanodrop). So I wonder if it worth it... I mean, I designed my primers and probes for real time so that they span intron-exon junctions to avoid genomic amplification. Is it not sufficient? Do I really need to digest the RNAs anyway? I know it is always better to do the more than the less, to get the most reliable results at the end. But if I lose in quality... what's the best solution??
I 've thought I could try to run one real time PCR with a raw RNA sample and a digested one to see if there is any difference in amplification. It could be an easy control as I don't have any genomic DNA to use as negative control.
Could someone help me, even in these hot sommer times (in Europe at least)?
thanks in advance
I don't digest with DNase and I don't have problems. Invitrogen says you should digest with DNase only if you are using primers that anneal in 1 exon, because you should have almost no contamination from DNA.
It;s hot in America as well.
If you want to be confident then you do try and divide a sample into two and DNase treat one aliquot and not the other and confirm you are getting same Ct values. I was always a little worried that if it was a small intron you may still get amplification (unless probe lay exactly on exon/intron boundary)
I do not DNase treat because I got the same results as you...poor yield, poor spec readings, poor Ct values, smeary bands on gel after treatment...I bought a couple different batches of 'rnase-free dnase' and I think they weren't entirely rnase free.
I run a certain % of controls (I check about 10% of my samples, but only with one primer pair so it is neither too expensive nor too cumbersome) on every plate where I amplify straight RNA sample as the control. All my primers are intron-spanning and I always check my melting curves (I use SYBR green). The frequency of gDNA contamination is pretty much nonexistent; I've found it in only 3 or 4 samples in 2 1/2 years of real-time, and on those occasions it was evident as well from strange Cts and also from melting curve doublets
anyways, I think it doesn't matter. I suppose different RNA extraction methods may vary, you should validate it with your method before proceeding, but I use Absolutely RNA miniprep kits and the gDNA contamination is pretty non-existent
this is just MHO...hope it helps
Thanks for your help, I will try to test one sample divided into 2 batches: with and without DNase and I will see...
I have thoroughly tested most commercially available DNase enzymes and I can tell you that only 2 were RNase free - Invitrogen's amplification grade and QIAGENs RNase free DNase set. All the rest caused significant RNA degradation in a liquid phase incubation. PRomega's wasn't too bad but it was nowhere near as efficient as the other 2
Have you tried Shrimp DNase?
In our hands it degrades less RNA than any of the "RNase-free" DNases, even if it's not marketed as RNase-free.
It do not degrade primers (ssDNA), and is easily heat-inactivated.
Gerd