Agilent 2100 Bioanalyzer used to check RNA quality
Photo courtesy: N. Garcia-Reyero.
Photo courtesy: N. Garcia-Reyero.
Sample Collection, Storage, and Handling
DNA or RNA must be extracted from a sample (e.g., water, or biological tissues) prior to analysis by PCR. Extraction methods will not be described here (see Life Technologies, 2012 for more information). Sample preservation, storage and extraction procedures should be documented to ensure that consistent procedures are used and that the results can be reproduced.
Evaluate DNA/RNA quality
- Purity of DNA/RNA can be checked by measuring the OD260/OD280 ratio with a spectrophotometer. Pure DNA and RNA have ratios of 1.8 to 2 (see Reardon, 2004).
- RNA integrity can be determined by running samples on a gel or on an Agilent Bioanalyzer to measure the ratio of 28S to 18S. Undegraded RNA samples have a 28S:18S ratio of 2 ( Reardon, 2004).
- RNA purity, e.g., the absence of inhibitors, can be evaluated using a technique called SPUD (Nolan et al., 2006). SPUD uses a template found only in potatoes and compares the amplification of the SPUD template in water with the SPUD template spiked into samples (Johnson et al., 2014).