I extracted viral RNA samples using Qiagen Viral RNA kit. the samples gave concentrations average 30 ng/ul and purity of average 2 for both 260/280 and 260/230. I shipped the samples using RNA transport buffer provided by a sequencing company. the QC report of shows similar results to mine using the nanodrop. But the RIN is very low in three samples ( 1.5 average) and the others are undetectable.
Is it possible to have good readings on Nanodrop and no results on bioanalyzer? I want to perform NGS on Illumina Hiseq, do you recommend proceeding with the samples or not?
Nanodrop quantifies the amount of nucleic acids present per unit volume but did not say anything about the quality/degree of degradation ofn it. Whereas, RIN calculation account for the degree of degradation and RIN algorithm calculates the quality and quantifies it. Basically similar to electrophoresis on gel, but gel cant be quantified accurately. RIN value is calculated with the help of capillary electrophoresis which help to quantify and access the quality of very small sample accurately. So Yes, it can be possible to get bad RIN number but no result is unlikely. There must be some result. For a good sequencing results, sample with good enough RIN (>7, in worst case 5) is a pre-requisite.
Thank you Abhijeet for your answer.
So the sequencing company want to proceed with samples as low as 1,5 in RIN do you recommend proceeding?
Also is there a difference in accuracy of Bioanalyzer when it comes to viral RNA?
Its always good to have high quality RNA to get exact expression profile. It would be good if you can have good quality RNA, if possible, otherwise you can go for the sequencing with current samples. If company agrees to sequence your sample, of course they can, but would be sure about the results and draw the conclusion you wanted to draw?
I haven't worked with viruses, so I cant answer that in particular. But as per the working principle, there should not be any differences in viral RNA or any other RNA in bioanalyzer.
RIN is calculated based on 28S and 18S rRNAs for eukaryotic samples (23S and 16S for prokaryotic samples). So it is not designed to evaluate viral RNA. As a result low RIN does not necessarily mean your viral RNA is of low quality. You have to look at the Bioanalyzer trace to decide for yourself whether your viral RNA is still intact. Your Nanodrop result is promising but it does not tell you whether your RNA is degraded or not, as degraded RNA can still have good 260/280 and 260/230 ratios. Bioanalyzer trace provides sizing info to help you make calls on whether there is degradation.
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