rna 260/280 ratio

Mrs R M D Lastrucci 075rlast at chiron.wits.ac.za
Thu Aug 24 07:51:21 EST 1995


Dear All

I am having problems with RNA extractions:  after lysing white blood cells 
in a guanidium-thiocynate containing solution,  2 phenol-chloroform 
extractions followed by a chloroform re-extraction, precipitation with 
isopropanol, wash with ethanol and resuspension in distilled water, I do a 
spec. reading at A=260nm and A=280nm.  According to the textbooks, a 260/280 
ratio of 2 with RNA is an indication of high purity; below that is 
indicative of protein contamination.  BUT, I am getting ratios in excess of 
2, almost as high as 3, and these samples definitely seem to be inhibited 
during PCR.  What does a >2 ratio mean?  Has anybody experienced this 
before, or know of a contaminant that absorbs at 260nm that could be a 
potential PCR inhibitor?  Re-extraction of the RNA after following the 
above procedure sometimes helps, but rarely takes the ratio down to 2. 

Thanks in advance for any help and suggestions

Rusla Lastrucci
Department of Haematology
University of the Witwatersrand Medical School
South Africa
Email: 075rlast at chiron.wits.ac.za
  



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