Dos
Do wear gloves and change them frequently.
RNases are secreted through the skin and skin contact will
contaminate your preps.
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-
contamination.
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'ts
Don't touch anything with bare hands
that will come into direct contact with RNA.
Don't breath 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.
(For more information see "Tips from the Bench: Effect
of Freeze-Thawing of Tissue on RNA Integrity"
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. |