Method to concentrate RNA - is speed vac method OK.. (Apr/03/2006 )

Hi All
I want total RNA and PolyA RNA for northern blots
I did not have the desirable concentrations of RNA after using Qiagen miniprep for my cell culture samples(primary, sceondary as well as cell lines)
for eg . I have 28 ug in 35uL ..
the procedure for northern blot wantd u to use 30ug RNA in a small voume say 2ul
wat would be the preferred method to concentrate the RNA that I already have
without worrying about degrading it
we are new to working with RNA..labmate suggested drying in speedvac...
wat do u say

please suggest ASAP
thanks a bunch

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Speedvac could work. Make sure to freeze your solution first on dry ice so you don't lose any when you vac it down.

The alternative is something like an isopropanol precipitation then resuspend in a small er volume. Likelihood is that you'll lose a significant percentage of the material you already have.

DNA is perfectly happy in a speedvac and I concentrate my DNA samples like that on a daily basis. Never tried it with RNA though. Might degrade, not sure.

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for alcohol precipitation, i use butanol for peletting procedure or etoh supplemented with glycogen.
washes are done in "standard" 70%etoh

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I think RNA to speed vac is nit so good, only for a short time at 37°C, not longer than 15 min, otherwise the RNA will degrade. we have made these experiences.
i would prefere for getting smaller volumes a precipetation with ethanol and resupend in TE or water.

Good luck

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Hi,
You can use ammonium acetate and glycogen for reprecepetation and after drying pelletes you can dissolve it in small volume.We are also using this methodes in our experiment hope it work well.
all the best.
awadh

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This is what I do to precipitate my mRNA. Add 0.1 volume of 3M sodium acetate and 1.0 volume of isopropanol and also 5 microliter of glycogen (20mg/ml). Incubate in overnight at -20 Celcius. Once u have the pellet after ultra speed centrifugation, juz air-drying it several minutes before redissolving it in water/EDTA should do.

I think the most important point is that you should add glycogen into it as it acts as a nucleic acid carrier in low RNA concentration which I think is ur case. At least for me, this solves the problem. Herring sperm DNA can also be used to substitute glycogen. Make sure your glycogen is RNase-free of course.

Hope this helps.

Regards,
Sang Ging

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