RNA isolation is both a skill and an art
Because RNA is prone to digestion by a wide variety of endogenous and exogenous RNases, and because these RNases are present on almost all objects that come into contact with humans, extreme care must be taken to prevent sample contamination and degradation. Diligence and adherence to some simple rules can make the difference between an intact, clean RNA prep and degraded RNA. The overall objective is to keep the mRNA profile of your sample intact for downstream applications in order to generate meaningful data.
Do decontaminate pipettors, gel boxes and gel combs. Treatment with RNaseZap™, RNase Decontamination Solution is a quick and thorough way to ensure that your equipment is free of all RNases.
Do use RNase-free reagents, tubes and tips.
Do process tissue quickly, by either disrupting in lysis buffer, freezing or storing in RNAlater™ Tissue Storage/RNA Stabilization Solution.
Do keep tissue frozen or in RNAlater prior to RNA isolation. This prevents breakdown of RNA by endogenous RNases and preserves the expression pattern of RNA species within the sample.
Do grind tissue that has been frozen on dry ice or in liquid nitrogen with a mortar and pestle. Following this, homogenize with a motorized homogenizer in lysis buffer. By keeping the tissue frozen, endogenous RNases remain inactivated during lysis. (For more information see 'Tips from the Bench: Effect of Freeze-Thawing of Tissue on RNA Integrity'
Do rinse your homogenizer in lysis buffer between samples to prevent cross-
Do be thorough in disruption and extraction. Tissue fragments left undisrupted represent RNA lost.
Do vortex for 2-plus minutes when phenol extracting. This will facilitate the removal of tightly bound proteins typically associated with RNA (these could inhibit downstream reactions).
Don't touch anything with bare hands that will come into direct contact with RNA.
Don't breathe on samples. Some researchers even wear masks.
Don't autoclave pipette tips, as water vapor in most autoclaves contains RNases.
Don't rinse tissue stored in RNAlater prior to processing.
Don't allow frozen tissue to thaw.
Don't resuspend RNA in DEPC water if the RNA is required for certain downstream applications. Residual DEPC can inhibit some enzymes and has been shown to adversely affect RT and translation reactions.