request advice on mRNA preparation

Robert Hewitt rhewitt at HELIX.NIH.GOV
Sat Oct 29 12:19:00 EST 1994


Dear Internetter,

I've been using a combination of the Stratagene micro RNA isolation 
kit and the 5'-3' spin column poly A selection kit, to make polyA RNA 
from human colon cancer cell lines.  My yields of mRNA are only 1-2% of 
the total starting RNA.  I'm told by the 5'-3' technical advisers, that 
these yields are on the low end of what you would expect for cell line 
RNA, and that you get higher yields with tissue RNA.  They also advise 
that you get higher yields if you poly A select from freshly prepared total 
RNA.  Can anyone suggest why this is?  Any comments or suggestions would 
be greatly appreciated. Thank you in advance,

Robert Hewitt




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