4.4. RNA quality assessment
The next step is to determine the condition of the RNA sample, prior to any assay. The three critical parameters are quantity, quality and integrity (i.e. absence of degradation). Quantity and quality are usually assessed by spectrophotometry (Green and Sambrook, 2012), by comparing the peak absorbance at 260nm (nucleic acids), 280nm (proteins) and 230nm (phenolic metabolites). A number of companies now market spectrophotometers and fluorometers that provide a complete UV absorbance profile from 1 μl of sample, from which the concentration of the nucleic acid can be determined, as well as its purity with respect to protein and metabolite contaminants. However, nucleic acid integrity can only be determined by running an electrophoretic trace profile, and assessing the degree of degradation by comparison of different nucleic acid size classes. The most comprehensive RNA quality analysis is through a chip-based microelectrophoresis system that provides a complete electrophoretic trace of the RNA sample which is used to quantify the integrity of the RNA, as well as the amount and purity (Bustin, 2000). Agilent, Qiagen, Invitrogen and BioRad market such systems. However, for fresh samples or those preserved with stabilizers or in a frozen transport chain, with little expected degradation, a simple UV absorbance spectrum is usually sufficient.
- Read the absorbance of an RNA sample at 230nm, 260nm and 280nm.
- A260 of 1.0 = 40 ng/μl
= 37 ng/μl ssDNA
= 50 ng/μl dsDNA
- A260/A280 < 2.0 indicates contamination with proteins.
- A260/A230 < 2.0 indicates contamination with phenolics.