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Old 05-14-2015, 01:20 AM   #3
rubyryan
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Location: New Zealand

Join Date: Sep 2012
Posts: 6
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Quote:
Originally Posted by Al Merry View Post
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!
Hi Al Merry, I have the same questions and so I was wondering if you used the speedvac to concentrate your samples and if it was successful and was also wondering if you were happy using the Turbo DNAse and if your arrays were subsequently sucesssful, many thanks Ruby
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