RNA degradation is a major problem with qPCR, as it introduces additional variability between samples that must be subsequently accounted for. Even if using equal quantities of RNA for each reverse transcription, differences in RNA quality may render samples incomparable without normalization (see Section 6.2 below). RNA quality may be adversely affected by tissue collection protocols as discussed above (see 'Light sensitivity' above), as well as freeze-thawing or improper storage, all of which give endogenous RNases the opportunity to degrade the RNA within the sample. Furthermore, the addition of exogenous RNases from contaminated plasticware and laboratory surfaces is all too easy due to the ubiquitous nature of these enzymes and their resilience to many forms of denaturation (Sambrook and Russell, 2001).
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