concentrating RNA - (Apr/22/2008 )
I have extracted RNA by qiagen column and i have eluted it by 30 uL (minimum elution volume) RNase free water. The problem is the low concentration (like 30ng/ul). I'd try to elute my sample twice by bigger volume and then precipitate RNA. Which is the best method to do that without losing and degradation RNA? I have to use RNA for qRT-PCR
Thanks a lot
If you are eluting in water you can just evaporate the water to concentrate, which is easier than precipitating the RNA.
A centrifugal evaporator would be best, but other approaches would also work.
I always concentrate it by evaporation. It is recommended to be done at 4ºC, but I always use room temp. and never had any degradation problems...
When extracting nucleic acid with those kits always use the maximum 50uL to elute them. You'll probably get more of the nucleic acid and if you need a more concentrated solution you can always concentrate it in some way.
I knew that but unfortunately we don't have equipment for evaporating!
i wouldn't recommend etoh precipitation, i lost half of my RNA when doing it (always blamed it on the pippets ). you could use the max volume in the elution, that way you extract more RNA, more diluted but more at the end. to concentrate it i used vaccum centrifuge. when doing the elutions you can do it by double, as the protocol suggests. to do the 2nd elution you can use your first elution and put it back in the column then spin again and hopefully get a higher RNA concentration.
i have found SC110 SPEEDVAC CONCENTRATOR!!!
It works at room T, 43°C and 65°C, so i'll use it at room T.
Do you guys think i'll have degradation problem in this way? I want evaporate about 100-150 ul of water, so how time it needs? I've never used it!
It should be fine, unless there is RNAse contamination. It will be fast, once the trap cools.
I always use the "micro" columns from Roche for small samples. There you can elute with only 10ul so that your RNA is already concentrated. It works pretty well also for fresh samples, not only for FFPE tissues.
The name of the kit is: High Pure RNA Paraffin Kit (Cat. 03270289001).
Price is even lower than Qiagen, however quality is about the same.
Maybe a dumb question, but what would happen if I just left it open on 65 degs C (never concentrated a sample before, so maybe it's just stupid), and wait till the water evaporates?
Apart from the obvious contamination issue (could do it in box), would the RNA degrade or something?
Use SpeedVac at room temp you could reduce the RNA volume by ~50ul in an hour. Or just sit it open in a laminar flow hood overnight and it will also reduce the volume by ~50ul.