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Old 05-17-2013, 10:13 AM   #1
Al Merry
Junior Member
Location: Oxford

Join Date: Feb 2013
Posts: 3
Talking Speed vac concentration of RNA?

Hi, I have many RNA samples extracted using Ambion Mirvana which I am planning to hybridise to Affymetrix HTA arrays. The protocol requires 150ng RNA in 3ul. My volumes currently range up to about 21ul, so I need to concentrate some of them.

Does anyone have any experience of using a Speedvac to concentrate small quantities of RNA? I've seen variable reports, but am loathe to use columns or precipitation as I fear losing more of my precious RNA in the process.

Also, has anyone ever used Biomatrica RNA concentrator or RNAstable that use anhydrobiosis (ie, drying) to stabilise RNA in a speedvac? It appears to act like a dessicant, but there's a publication comparing microrarray analysis of samples before and after Biomatrica concentration that was unable to detect any difference caused by the process (Hernandez et al Biotechniques 2009 Vol 47pp667-670).

I know this is a sequencing forum, and I have prepared DNAsed samples of the RNA for sequencing, but for the microarray, would anyone care to comment whether it is fine to use the pre-DNAse-treated RNA samples, having determined RNA concentration by Qubit assay?

Finally, I used a Qubit dsDNA HS assay and an RNA assay to determine the RNA and DNA concentrations before and after DNAsing using Ambion Turbo DNAse and purifying using Qiagen cleanup columns. I was discouraged to find that after DNAsing, I had a higher DNA:RNA ratio. Has anyone ever used such an assay to determine the effectiveness of their DNAse treatment? The DNAse batch that I was using was a few months old, in fact I finished the tube.

Thanks very much for your help!
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