8.4. Nucleic acid quality assessment
A number of sophisticated methods are available to determine the quantity, quality and integrity of an RNA sample, and described in the BEEBOOK paper on molecular methods (Evans et al., 2013). The minimum requirements are to determine the yield and purity of the RNA with respect to protein and phenolic metabolite contaminants. This can be determined by UV spectrophotometry (Green and Sambrook, 2012), through comparing peak absorbance at 260nm (nucleic acids), 280nm (proteins) and 230nm (phenolic metabolites):
- A260 of
1,0 = 40 ng/μl ssRNA
= 37 ng/μl ssDNA
= 50 ng/μl dsDNA
- A260/A280 < 2.0 indicates contamination with proteins.
- A260/A230 < 2.0 indicates contamination with phenolics.