-
Assessing RNA quality
- Chemical impurities such as detergents, denaturants, chelating agents and high concentrations of salts should be avoided as these may affect the efficiency of enzymatic steps.
- Other contaminants such as DNA, proteins and dyes may also reduce the efficiency of steps in the library preparation.
- The quality of RNA may be assessed by Nanodrop (for samples with concentration >20 ng/µl).
- We recommend that sample RNA has a 260/280 ~ 2.0 and a 260/230 ~2.0-2.2.
- A 260/280 which is lower than ~2.0 indicates the presence of DNA.
- A 260/280 which is lower than ~1.8 can indicate the presence of protein or phenol.
- If the 260/230 is significantly lower than 2.0-2.2, this indicates the presence of contaminants, and the RNA may need additional purification.
- Use the Agilent Bioanalyzer together with RNA Analysis Kits to assess whether the RNA has degraded.
The Bioanalyzer traces below show an example of a good quality, full-length RNA sample (top) with an RNA Integrity Number (RIN) value of 9.5, and a degraded sample (bottom) with a RIN value of 4.5. For more information about RIN values, please see the RNA Integrity Number page in the Know-How section of the Community.