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Old 05-17-2013, 09:13 AM   #1
Al Merry
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Location: Oxford

Join Date: Feb 2013
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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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Old 05-17-2013, 04:59 PM   #2
JamesH
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I've never done it (speedivac RNA) but we have a speedivac in the lab and I've seen a few people try, mostly without much success (the RNA tends to end up degraded).
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Old 05-14-2015, 01:20 AM   #3
rubyryan
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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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Old 05-14-2015, 01:48 AM   #4
Al Merry
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Hi Ruby, I decided against using the speedvac as I was wary about RNAse contamination. I was very happy using the Turbo DNAse. After tweaking the procedure, I ended up DNAsing using Turbo DNAse, then cleaning up with Qiagen minelute columns (using a few precautions to enhance purity), then precipitating in the presence of LPA as carrier. Recovery and quality were good, paper is submitted. Detailed method is:
DNAse up to 20ug in 100ul using 1ul DNAse according to Ambion Turbo DNAse instructions. Don't add stop solution after 37 degree incubation for 20 minutes, but proceed directly to next step (Qiagen Minelute cleanup). DON'T put on ice! (many forum posts cautioning against this)
Add 3.5 vols warmed RLT (@37 degrees; 175ul to 50 ul, 350ul to 100ul)
Add 1.5 vols EtOH (350ul if 50ul+175ul, 700ul if 100ul +350ul)
Add to minelute column
Spin 15s top speed
Wash 500ul RPE thoroughly (2mins, or roll - I found lots of forum posts stressing the importance of washing the rim of the column, to wash away all the salt which could be carried through as impurity later on). Spin
Wash 500ul 80% EtOH thoroughly. Spin
Spin, lid off, 5 mins, top speed
Elute with 14ul H2O. RT 5-10', spin 1' top speed.
I then needed to precipitate to concentrate my RNA:
1ul Linear Polyacrylamide (Sigma)
10ul NH4OAc
75ul H2O (can prepare these three as a mix and add 86ul)
250ul EtOH
-80 degrees 15 mins or -20, 3 hours
spin 4 degrees, 10'
2x wash 70% EtOH, RT 5', Spin 4 degrees, 5'.
Dry 55 degrees 5'
Resuspend in a suitable volume (5 or 10ul for me).

Hope that's interesting, good luck!

Al
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Old 05-14-2015, 02:47 PM   #5
rubyryan
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Many thanks Al, thats very helpful and I may try this approach, kind regards, Ruby
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