Ribonucleases (RNases) are very stable and active enzymes that degrade RNA. Minute amounts are sufficient to destroy RNA. It is important to use Rnase free conditions during all apsects of tissue collection.
Sterile technique should always be used when working with RNA. Always wear latex or vinyl goves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed.
Disposable Pastic ware:
The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNAse-free and do not require pretreatment to inactivate RNases.
Non-disposable plastic ware should be treated before use to ensure that it is RNase free. Rinse thoroughly with 0.1M NaOH, 1 mM EDTA followed by RNase-free water.
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with detergent, thoroughly rinsed and oven baked a * 240°C for 4 or more hours (overnight, if more convenient) before use. Autoclaving alone will not fully inactivate many RNases. Oven baking will both inactivate RNases and ensure that no other nucleic acids are left on the glassware surface. Alternatively, glassware can be treated with DEPC* (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC, allow to stand overnight (12 h) at 37°C, and then autoclave or heat to 100°C for 15 min to remove residual DEPC.
*DEPC is a suspected carcinogen.
Metal instruments (scissors, pick-ups, etc) can be autoclaved prior to use or washed with DEPC water and baked as described above under glassware.
Alternatively, all of the above, plastic and glass ware, instruments and work surfaces may be treated with RNase Zapô, a RNase decontamination solution. This product is made by Ambion (see below for order information).
Sample Collection and Storage
Rodent or other Non-human samples:
At necropsy, tissues should be collected within 12-15 minutes following death. No specific form of euthanasia is required. If the tissue collection time is much greater than 15 minutes, RNases will have been activated and it is highly unlikey that good quality RNA will be obtained for further analysis.
Remove the tissue of interest and quickly cut into cubes no larger than 3 mm. The cubes are immediately transferred directly into a plastic weigh boat containing liquid nitrogen. After the tissue has been completely frozen, use clean pick-ups to transfer the tissue into a sterile cryovial . The cyrovial is then placed in a liquid nitrogen container. Do not over load the cryovial or it could possibly burst. The samples are stored at -70°C. Most RNA isolation kits guarantee their kits to extract quality RNA from tissue samples that have been stored for 1 year at -70°C. If you plan on long term storage of your samples, store them at -130°C (in liquid nitrogen). Most degredgation reactions are stopped at -130°C. Do not allow the samples to thaw during the transfer to the freezer. The vials can be removed from the liquid nitrogen container, placed on dry ice, organized and stored in a carboard cryovial box and then placed in the -70°C freezer.
Instruments should be cleaned between tissues and animals by dipping in 70% ethanol. Wipe the instruments with a clean Kimwipe before using again. Weigh boats and blades should be disposed of after each sample. The ethanol should be changed between each animal.
Important: If there is a defined target tissue to be collected in a study, collect this tissue first in a timely manner. In the interest of time, it is advisable to have a separate frozen tissue collection area set up in the same room or area as the necropsy. This allows for the tissue to be collected and processed in a sterile manner and timely fashion without disrupting the remaining tissue collection during the necropsy.
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