Appendix B: General Remarks on Handling RNA
Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after purification. To create and maintain an RNase-free environment, the following precautions must be taken during pretreatment, as well as using disposable and non-disposable vessels and solutions while working with RNA.
General handling
Proper microbiological and aseptic techniques should always be used when working with RNA. Hands and dust particles may carry bacteria and molds, and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the skin’s surface or dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice when aliquots are pipetted for downstream applications.
Disposable plasticware
It is recommended that sterile, disposable polypropylene tubes be used throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.
Non-disposable plasticware
Non-disposable plasticware should be treated before use to ensure that it is RNase-free, which consists of thoroughly rinsing plasticware with 0.1 M NaOH and 1 mM EDTA,* followed by RNase-free water. Alternatively, chloroform-resistant plasticware can be rinsed with chloroform* to inactivate RNases.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent, thoroughly rinsed, and baked at 240°C for at least 4 hrs or overnight, if more convenient, before use. Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethylpyrocarbonate); fill glassware with 0.1% DEPC (0.1% in water), allow it to stand overnight (12 h) at 37°C, and then autoclave or heat to 100°C for 15 min to eliminate residual DEPC.
Electrophoresis tanks
Electrophoresis tanks should be cleaned with a detergent solution (e.g., 0.5% SDS),* thoroughly rinsed with RNase-free water followed by ethanol,† and allowed to dry.
Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong but not absolute inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware, or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. To treat solutions, add 0.1 ml of DEPC to 100 ml of the solution and shake vigorously to bring the DEPC into the solution. Let the solution incubate for 12 h at 37°C. Autoclave for 15 min to remove any trace of DEPC. DEPC reacts with primary amines.
*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. Consult the product supplier’s safety data sheets (SDSs) for more information.
†Plastics used for some electrophoresis tanks are not resistant to ethanol.
*Take proper care and check the supplier’s instructions; it cannot be used directly to treat Tris buffers. In the presence of Tris buffers, DEPC is highly unstable and decomposes rapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC can modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated with extremely low efficiency in cell-free systems. However, its ability to form a DNA:RNA or RNA:RNA hybrid is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must be eliminated from solutions or vessels by autoclaving or heating to 100°C for 15 min. Note: RNeasy buffers are guaranteed RNase-free without DEPC treatment and, therefore, free of DEPC contamination.
From RNeasy Plant Mini Handbook*
*RNeasy Plant Mini Kit. [Accessed 2023 February 23]. https://www.qiagen.com/fr/resources/download.aspx?id=246847e7-0095-43e4-8d1d-41df3f9153dd&lang=en
