Bioanalyzer for viral RNA samples

[Smriti]

Hi There,

Has anyone used Bioanalyzer Pico/Nano RNA assays for viral RNA analysis? I am new to the viral genome analysis. I have tried running both the kits with Eukaryotic total RNA assays and haven't seen much luck there. If bioanalyzer is not the right choice, whats the most common tool used to test viral RNA size and integrity?

Thanks!

[tomdriedonks]

I use Bioanalyzer Pico for quantifying purified smallRNA from extracellular vesicles. These have roughly the size of viruses (around 100 nm diameter) so I suppose RNA content would be comparable to your situation. Bioanalyzer Pico is pretty good in qualitative measurements, quantitation of RNA (most problematic in the 100 pg/ul - 1000 pg/ul range) shows variation. But from your question I understand you're mostly interested in sizing, so you will probably be OK. In case you have a very small RNA genome, Bioanalyzer small chip might be something for you.

Note that Bioanalyzer is optimized for total cellular RNA analyses; RNA integrity is estimated as the ratio of 18S / 28S RNA peaks. So it will probably tell you your viral RNA samples are not intact. However, as you know the size of the viral RNA genome I'm sure you can make an estimation on the quality of your own RNA samples.

[Smriti]

Hi There,

Thanks for your suggestions! I am using Prokaryotic total RNA Pico assay for analyzing quality of RNA extracted from virus, right after extraction. Probably, its low viral loads and I am expecting extracted RNA size to be approx. 9kb.

Do you think they calculate RIN scores based on ribosomal bands for prokaryote assay too?

[tomdriedonks]

Hi Smitri,

Bioanalyzer RNA ladder runs up to 6 kb, so it could be your viral RNA is too long.
I have no experience on this since my research topic focuses on small RNAs (<300nt), but you could check with Agilent whether it is technically possible to detect RNAs of this size.
About your concern of having too low RNA concentrations; Bioanalyzer Pico will still detect RNA at very low concentrations (lower than 100 pg/ul) but quantification is not very accurate. Do you have other means to quantify your extracted RNA?