Is it possible to view mRNA on gel - (Oct/17/2008 )
Iam new to work on RNA
I use poly A tract mRNA isolation system to isolate mRNA but unable to view mRNA on gel.
any suggestions are requested
mrna will appear as a smear or cloudiness in the lane, not as discrete bands, but yes, you can see it on a gel if you load enough.
Hey novagene have a look at this thread, i hope it could help you .
I have isolated mRNA and precipitated using sodium acetate and isopropanol , stored at -20 overnight ,spin at 10000 for 30 min. and used it for synthsizing cDNA but could not observe any bands on gel. awaiting for suggestions.
After cDNA synthesis, performe PCR. Then you should be able to see band(s). M.
I will definately try out and get back again.
I did PCR ,but no band . it seems I am loosing the mRNA while precipitation. any best method for mRNa ppt. my experiment insists on containing 5micrograms of mRNA in 4microlitres.how to go along with it ??????
Thanks a lotttttttt
5 ug? That's a lot... I usually make my cDNA synthesis with 1 ug RNA in 20 ul of total mix... You know, buffer, MgCl, dNTP, primers, water, rev. trans. RNaze inhibitor... After that I take 5 ul for PCR (in two step protocol which I prefer).
Are you using random primers or specific primer for cDNA synthesis? And what about protocol, denaturation, annealing temperature, DNA synthesis temperature? Too much of template is as bad as too little .
For start, after RNA extraction I would make gel electrophoresis to see if I have and RNA in my sample. Also, usually add sterile water rather than TE buffer. And I precipitate RNA the same way as DNA (1/10 NaOAc, 2 V 100% EtOH). After 30' in -80, or O/N -20 and centrifugation at 15 000 x g, I decant supernatant, add 70 % EtOH, and precipitate again as before, centrifugation 15 000 x g, decant and dry pellet at RT for 30'. Be sure that no EtOH is present after that (smell your sample). Also, be sure that you don't have RNase contamination in your solutions. M.
I had problem with RNA extraction and cDNA amplification as well. Problem is I can see the smear on the gel after total RNA extraction. But I failed in first strand cDNA synthesis. I do not know where the problem is. Because I may lose the specific mRNA during the extraction since I want to amplify specific gene.
Hi AS mikkel,
perform RT_PCR with the internal control primer, then u will know wheather your 1 strand is synthesized or not.