RNase treat gDNA?

[nohara]

Hi all,
I am preparing gDNA for Illumina sequening (short reads and paired-end). I will be quantifying the sample using qPCR. I am wondering why RNase treatment is recommended in some sample protocols and if I really need to do it. Could it potentially interfere with quantification by the genomics facility where I am sequencing? I would prefer not to RNase treat as I have limited DNA and I don't want to mess with it too much. DNA was extracted using a Qiagen kit and looks pretty pure. Could small amounts of RNA disrupt the library preparation process or the quantification process when I send the samples in for library prep?
Any help would be appreciated!

[pmiguel]

Hi all,
I am preparing gDNA for Illumina sequening (short reads and paired-end). I will be quantifying the sample using qPCR. I am wondering why RNase treatment is recommended in some sample protocols and if I really need to do it. Could it potentially interfere with quantification by the genomics facility where I am sequencing? I would prefer not to RNase treat as I have limited DNA and I don't want to mess with it too much. DNA was extracted using a Qiagen kit and looks pretty pure. Could small amounts of RNA disrupt the library preparation process or the quantification process when I send the samples in for library prep?
Any help would be appreciated!

A typical genomic DNA prep is >90% RNA. Qiagen can do better than that, but probably does have a good deal of RNA in it. So, the first problem with RNA is that it throws off UV spectrometric estimates of the concentration of RNA. But if you are using some other method (agarose gel or fluorimetry) then RNA is not a quantification issue.

Second, will T4 DNA ligase ligate extraneous RNA to your DNA adapters? If it can, then you want to reduce the amount of RNA in your sample as much as possible to get optimum results. A decade ago I would have said "no way". But now I am less certain. There is an excellent paper on the topic: Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochemical Journal 398: 135-144

I thought I had posted in detail about this before. But google is showing me little. So I whipped up a post and put it here.

Anyway, to a first approximation, a little RNA is unlikely to cause problems, but with DNA preps there is often quite a bit more than "a little" RNA present.

--
Phillip