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I extracted RNA from different cell lines, an I want to perform reverse transcription and then PCR. To get good results, in which range should the absorption ratios 260/280 nm and 260/230 nm be? And what is the idea behind measuring those ratios?

I got values for the 260/280 ratio from 2.1 to 2.13 and values for the 260/230 ratio from 0.98 to 2.01. Are these values acceptable, and what information can I gain from them?

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up vote 9 down vote accepted

DNA and RNA absorb at 260nm. Proteins absorb at 280nm. The 260/280 ratio is a good estimate of how pure your sample is. For RNA, the 260/280 should be around 2. If it is lower, this might be an indication from contamination or proteins, phenol, or other contaminants in your sample. The 260/230 ratio is a second measure for purity of the sample, as the contaminants absorb at 230nm (like EDTA). The 260/230 ratio should be higher than the 260/280 ratio, as it is usually between 2 and 2.2. Lower ratio might be an indication of contamination.

In your case, I would be worried about the purity of the sample which gave you 260/230 ratio of 0.98. If you are using nanodrop, looking at the plot can also be a good indication of the quality of your sample. For a pure sample, a well defined peak (no shoulders or wiggles) at 260nm is expected.

Take a look at the NanoDrop manual for more information.

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Indeed I remember than the sample with the ratio 260/230 had two peaks. In my case the values for the 260/230 ratio are never higher than the 260/280 which indicate I have contamination. What can be the consequences not to have the correct values for these ratio for a PCR? I will extract RNA soon. How can I prevent a contamination next time? What is the good practice? –  kali281 Jan 12 '12 at 8:57
    
If you have two peaks is a clear indication of contamination. Don't look only at the ratios because this gives you some idea, but doesn't make a strong statement. The PCR might fail due to contamination but you might try it anyway. You might also try isolating your RNA again. –  Gergana Vandova Jan 12 '12 at 9:05
    
For the sake of completeness and relevant detail: The aromatic amino acids (Phe, Trp and Tyr) are the primary contributors to the ability of proteins to absorb at 280nm. Cysteine double bonds can also absorb but its both rare and the extinction coefficient is smaller in comparison to the aromatic residues. –  gkadam Aug 6 '12 at 2:16

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