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Isolation of protein and RNA from RNeasy Minikits?

rna western blotting protein extraction RNeasy

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5 replies to this topic

#1 jac05021

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Posted 06 May 2013 - 08:02 AM

Hey everyone,

So I'm trying to see if I can isolate protein and RNA from the RNeasy Minikit. I am working with about 7 mg of rat nasal epithelial tissues and therefore need an efficient way to get both RNA and proteins from the same small sample. I know they sell a DNA/RNA/protein kit from Qiagen, but it is very expensive. TRIzol is also not going to be an option because I have not had good luck isolating RNA from my samples with their methods (contamination issues and low RNA yields).

Any suggestions on how I can isolate both total protein and RNA from my rat samples using this RNeasy method (or one just as costly...or cheaper :-) ).

Thanks!

Joe

#2 pDNA

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Posted 06 May 2013 - 10:19 AM

do you tried this one (uses Trizol):
[url="http://www.ncbi.nlm.nih.gov/pubmed/17489233"][url]http://www.ncbi.nlm.nih.gov/pubmed/17489233[/url][/url]

concerning the RNeasy columns ...you purify the RNA using the column and then you precipitate the flow through, like here:
[url="http://www.biotechniques.com/multimedia/archive/00005/BTN_A_06401BM05_O_5037a.pdf"][url]http://www.biotechniques.com/multimedia/archive/00005/BTN_A_06401BM05_O_5037a.pdf[/url][/url]

Precipitation is also described [url="http://www.google.at/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CEMQFjAB&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B7B79FE96-71A3-467F-AA8B-767A9DE6F200%257D%26lang%3Den%26ver%3D1&ei=JPOHUceJDoeMswb58oGACQ&usg=AFQjCNEyd7b1C68nkMRIDopKbtpjyykY_g&sig2=76CAnishOLJk96oUS-e5_g&bvm=bv.45960087,d.Yms&cad=rja"]here[/url].

Regards,
p

#3 jac05021

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Posted 06 May 2013 - 12:24 PM

Thanks! The RNeasy column protocol will be the one i try next! thanks

#4 jac05021

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Posted 10 May 2013 - 12:56 PM

by the way, this protocol worked beautifully. I did make some editions though.

I find it better to add 1.5 volumes of IPA to the 1st flow through from the RNeasy column (ie from the 70% EtOH addition step). I then continue with the protocol verbatum. It saves on having to wait a day of letting your tubes sit at -20C. I was able to get RNA and whole cell lysate protein samples from the same 4-5mg chunk of nasal tissue!

Thanks again.

Joe

#5 GHANBAR

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Posted 11 May 2013 - 01:22 AM

hello

I need protocol RNA extraction that cheap and have good qualify.

THANK you

#6 pDNA

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Posted 12 May 2013 - 08:55 AM

[url="http://openwetware.org/wiki/RNA_extraction_using_trizol/tri"][url]http://openwetware.org/wiki/RNA_extraction_using_trizol/tri[/url][/url]

Best,
p





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