RNA extraction, RT-PCR problems

Jose de las Heras josenet at tiscali.co.uk
Tue Apr 25 05:21:19 EST 2006


Long shot, but...

you say you use random hexamers for your RT. Do you use total RNA as it 
comes from the Trizol extraction or do you purify to mRNA first? If you use 
total RNA, only about 1-3% of the RNA you measure will be mRNA, the rest is 
ribosomal, mainly. Could it be that you had much less RNA for those samples? 
If you start with very little mRNA in your sample, and perhaps there's also 
some degradation, the effect could be big.
Why don't you use an oligo(dT) primer, if you use total RNA?

I use "anchored" (dT)20 (20 "T" plus random A/C/or G, plus A/G/C or T 
(22-mers) for my RTs, for cDNA labelling, etc... and it works pretty well. 
Just don't buy the oligos from a commercial source, they charge you a lot. I 
order mine from our favourite source of synthetic oligos, and choose their 
purest option. I get TONS of the stuff for less than £30.

Jose 




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