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Handling RNA - How to avoid RNase contamination?



Hi Susan,

I would second what tea-test has pointed out. Any tissue you process is going to be loaded with RNases (cells contain RNases). Any residual RNase present on your homogenizer is going to be very minor compared to what is already present in your cells. All you need to do is wash your homogenizer with soap and water and give it a rinse with clean MilliQ or DEPC water.

In my experience working with RNA, the majority of the degradation happens at the initial stages of the isolation. You need to work quickly to get the cells lysed and homogenized within whatever lysis buffer you are using. Sometimes RNA degradation is a problem for very inexperienced users who are very slopping (touching the insides of tubes/lids with fingers even with gloves on, leaving lids open so that bacteria (that contains RNases) can float in, taking way too much of the top layer and getting the white goop (protein) when using Trizol etc.). That not withstanding, your biggest challenge will be to get the tissue homogenized quickly.

A note on RNases: Autoclaving is not effective at eliminating RNases. Check:
http://www.ambion.co.../tb/tb_178.html

Also, even if autoclaving destroyed RNases, it would not be necessary to do. As tea-test pointed out, there should be very little RNases on your homogenizer, compared to what is present in your cells so autoclaving is not helpful here. Don't worry about using RNase Zap or any of that stuff on your homogenizer.

Another tip: When I do RNA work, I make sure I start with sterile/RNase free tubes. I take a whole bag and dump it out in the flow hood (not fume hood). Put on a clean pair of gloves. Cap all the tubes and store them capped in a jar. Now you have a clean supply of tubes ready to be used for any application, including RNA work. If you don't do this you may find yourself touching the inside of the caps when you close them when you go to label them. Your gloves will pick up RNases from the environment so don't consider them RNase free when you touch things.

6 Comments

I extract RNA from patients samples for detection of human viruses. We kept the samples in lysis-buffer (Qiagen RNA extraction kit) then later carry out extraction. Do you think it is a bad practice? Should we work immediately after adding samples to lysis buffer?

Thanks.
What is the sample? It all spends on if the lysis buffer is getting into the tissue/cells to protect the RNA from degradation. If it is tissue biopsy, the inner tissue that is not in contact with lysis buffer will have RNA degradation, that is why it is recommended to mince with scissors to less than 0.5mm thickness so that the sample can not only be coated in buffer, but it should be able to get to the inside portion of your sample.

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