RNA HYGIENE (PART IV) – Safeguarding RNA Integrity
Since RNA is so chemically fragile, working with the molecule can feel a bit like skydiving without a parachute. While we’ve previously discussed protecting RNA using ribonuclease inhibitor proteins (RIs), RIs have very limited utility and use due to their own fragility. So, are there other more robust measures that can be implemented to guard the safety and integrity of RNA during routine manipulation? Fortunately, the answer is yes.
Protection of RNA begins with protection of the biological sample itself. The sample containing your RNA of interest is usually chock full of RNases. One way to protect your RNA is to flash freeze the sample at the time of collection thereby reducing RNase activity. Also, numerous RNA stabilization reagents are commercially available that can be added directly to a sample to inhibit RNase activity prior to RNA purification. These protectants penetrate cells and can even be added to flash frozen samples to protect RNA during the thawing process.
When working with RNA, another safeguard is to keep all materials on ice. This includes all reagents and even the water you are using. The activity of any residual RNases in reagents and sample RNA will be greatly reduced. As an added bonus, colder temperatures also reduce degradation of RI proteins (see previous blog). Also, be sure to minimize the amount of time samples or reagents are not on ice during manipulations.
The type of water used in RNA manipulation is often misperceived as insignificant; however, in reality it plays a huge role in RNA protection. Whether it be for sample dilution or reaction addition, water is the universal solution. But even water can contain RNases. Water marketed for RNA use is often treated with a chemical called diethylpyrocarbonate (DEPC). And while DEPC is a potent inhibitor of RNases, it is not without its issues. For example, DEPC in RNA samples can cause inhibition of some downstream enzymatic processes (such as in vitro transcription). Further, DEPC is rapidly degraded in buffers like Tris and HEPES making it essentially useless in protecting RNA. But there is good news — water that is ultra-filtered and certified RNase-free but NOT DEPC treated is also available and often more beneficial.
On a related note, DEPC can also be used for glassware treatment to make it safe for RNA. Following treatment, the glassware should be heated and dried prior to use. In the long view, however, it may be more convenient to use sterile disposable plasticware certified RNase-free instead.
While taking the leap to work with RNA can seem somewhat precarious and daunting, the extra precaution and care you take will always make sure you have a safety net to catch you!