Determining quality of polyA+ RNA

CK Wen cwen at aux.btny.purdue.edu
Mon Oct 30 11:07:27 EST 1995


Subject: 
              Determining quality of polyA+ RNA
        Date: 
              Sun, 29 Oct 1995 22:54:07 +0900
       From: 
              lamphier at po.infosphere.or.jp (Marc Lamphier)
Organization: 
              University of Tokyo
 Newsgroups: 
              bionet.molbio.methds-reagnts 


The ratio of 28S:18S rRNA is a good indicator to determine the quality 
of RNA preparation because the molecular weight ratio of 28S:18S rRNA is
about 2:1 (4Kb:2Kb).  After oligo(dT) enrichment for poly(A)RNA, there 
is still rRNA carrier-over, and the poly(A)RNA mw is most abundant at 
the position around 1-2Kb.  Plus the carrier-over of 18S rRNA, we should
expect to see a stronger band at the position of 18S rRNA.  Remember, 
the poly(A)RNA composes of 1-2% of the total RNA, and after enrichment, 
2KB is the position where EtBr gives the strongest signal.  

To determine the quality of poly(A)RNA, you may want to do in vitro 
translation or a Northern hybridization.  For the Northern, I would like 
to use a specific cDNA or genomic clone as a probe which shall give a
single band.  If you use oligo(dT), it only shows you that your poly(A)
RNA has a poly(A) tail and would not tell you if the RNA has a good 
quality.  Using Church buffer is a good choice which gives less 
background and only needs one washing buffer.

One question about your suggestion of using the FastTrack.  Based on the
assumption that poly(A)RNA composes of 1-25 of total RNA. You mentioned
that the FastTrack gives 4-5X poly(A)RNA yield. Have you ever measured
how many percent of your resulting poly(A)RNA composes of in the total 
RNA?  I do not know very about the FastTrack. However,if it's a "batch
method" to enrich the poly(A)RNA, the carrier-over of non-poly(A)RNA is 
much higher, because the olig(dT)-slurry has a certain volumn which may
trap non-poly(A)RNA and it looks like that a higher yield is obtained.
It's why chromotography and the paramagnetic particles give less RNA 
than the "batch-method".  However, the carrier-over is less in the 
former methods.

Chi-Kuang Wen





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