mirVana kit - I can't have small RNA isolated!!! (Sep/23/2009 )

Hi

I'm using mirVana miRNA isolation kit. I had total RNA from 9 different lymphoid cell lines and I used the protocol FII to enrich for small RNAs - after total RNA extraction with trizol. After that, I analysed the purity of RNA in the Agilent 2100 Bioanalyzer, and for all cell lines, except one, I got a rRNA contamination between 6-15%, witch is too much for my proposes. In the only sample that I got 0% of contamination, the yield was too low (7ng/ul in 50ul).

then I also tried the additional procedure IV.A.. This last one I tried twice in the same samples and still I get contamination (the results are the one in attachment). At this time I already spend one entire isolation kit and I still don't have the purified small RNA fraction, and I don't know what I should try more with this kit!

can someone help me?

tks

In your case, I will suggest you to take one sample and extract total RNA by using trizol. Then mix your RNA with binding buffer (in any miRNA isolation kit) and add ethanol to final concentration 10, 20, 30.....70%, go through the first column (the large RNA bind to the column), recover the "flow-through" to see which one gives you "pure" small RNA.

The whole idea is to use ethanol% to separate RNAs, small RNA binds to the column when ethanol concentration is high.

Dear Nadia,

We also use this kit to isolate small RNAs and the kit works fine. What experiment will you carry out after isolating the small RNAs? I have asked Ambion before...the enrichment step is for experiment like northern blot. if you just want to do real-time PCR to quantitate the RNA level, I think you shoud use teh protocol F.I. to isolate TOTAL RNA.

Samantha

Thank you both for your help

I will try that with increasing ethanol concentrations, and choose the best one.

Answering to Samantha, I want to use the RNA for microarrays (and for that I really need to enrich for small species) and also for q-PCR. The problem is that with total RNA i got non specific amplification in the real time, so I'm trying to enrich the RNA in small species to see if I can avoid that inespecificity. I'm using trizol to extract total RNA and then I use the IV additional procedure, because with FII didn't work.

tks

Nádia

FYI, you may get 300-500ng small RNA (less than 200nt) by using whichever miRNA purification kits from 10ug total RNA extracted from Trizol.

Nádia.c on Oct 14 2009, 08:20 PM said:

Hi, i've seen your post just now and i confess i do not understand which kind of problems u r experiencing.
what do you mean when u say that u have non specific amplification in real time? are speaking of miRNAs or what? if yes, how do u amplify miRNAs? I ask that because i extract total RNA from many different samples with different kits (including miRVANA) and trizol but i NEVER experienced any kind of contamination or aspecific amplification. are u sure the problem is in extraction and not in what u use to amplify miRNAs?