Unlike DNA, which can last for eons, RNA is a fragile and degradation-prone cousin. After working with RNA for a while, one becomes quite paranoid about handling RNA because even a single sneeze or drop of saliva can potentially affect your results. The reason is that there are enzymes called RNases that specifically target and degrade RNA. In this article, I will discuss five mistakes that will no doubt hype your paranoia about the possible ways to screw up your RNA prep.
1. Not keeping the working environment RNase-free
The most common source of RNase contamination is actually your own skin. The surest way to spread it is by touching different working surfaces with your bare fingers. One of the best ways to prevent RNase contamination is to maintain an RNase-free environment. Wear gloves at all times and change them frequently. Also, make sure to clean up the workspace with RNaseZap and/or UV light before and after each prep.
2. Not using RNase-free water
It is important to only use RNase-free reagents such as diethyl pyrocarbonate (DEPC)-treated water and RNase-free labware.
3. No ice, no dice
There is a reason why all RNA preps are done on ice. This is because RNases function optimally at room temperature – keeping all your samples and reagents on ice will ensure that you prevent RNA degradation.
4. Not using filter tips
It is important to use RNase-free tips that contain a filter. Otherwise, it is very easy to spread contamination from one sample to another. Filter tips block any carry-over and prevent spread between samples
5. Not having a dedicated set of pipettors and tubes reserved for RNA application
It is important to keep labware and reagents used for RNA preps separate and reserved specifically for that purpose. Otherwise, it is very difficult to ensure that these instruments, especially pipettors, are RNase-free.
Hopefully these five examples of what not to do will help you avoid messing up your RNA preps.
In my next article I will discuss how to troubleshoot your RNA isolation in case something does go wrong. Stay tuned!