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Handling RNA - How to avoid RNase contamination - (Nov/24/2009 )

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Hi,

I'm doing MRNA isolation from tissue, using rotar-stator homogenizers. So i'll put the metallic end of the this "mixer" in my tissue sample. Because it is important to keep everything RNase-free, i wondered how i can make the metallic end, RNase-free?

greetz,

susan

-susanna-

Hi susanna, You can autoclave the metallic pestle and wipe all the surfaces coming contact to the tissue with RNaseZap. Don't worry If you dont have RNaseZap in the lab, Just make sure you homogenize the tissue soon and always keep it cold and add the lysis buffer ASAP after grinding. This should do the job!

-gogreen-

gogreen on Nov 24 2009, 08:05 AM said:

Hi susanna, You can autoclave the metallic pestle and wipe all the surfaces coming contact to the tissue with RNaseZap. Don't worry If you dont have RNaseZap in the lab, Just make sure you homogenize the tissue soon and always keep it cold and add the lysis buffer ASAP after grinding. This should do the job!



Can i do autoclaving, for electronic devices like rotar-stator homogenizers?
Can i also just clean the metallic tip of the device with the RNasezap?

greets
susan

-susanna-

susanna on Nov 24 2009, 11:03 AM said:

Can i do autoclaving, for electronic devices like rotar-stator homogenizers?
Can i also just clean the metallic tip of the device with the RNasezap?

greets
susan


does the probe come off? if so, then you can autoclave the probe.

rnasezap alone should be okay.

-mdfenko-

Wipe clean the probes and cleaning with RNaseZap should do...As mdfenko said, the probes can be autoclaved if they can be removed from the machine

-gogreen-

I would say that the tissue samples from which you are isolating RNA per se are not RNase free, so the question is if those spurious amounts of RNase that might be on your extraction device will have an impact on your results. I would have more concerns about cross-contaminating your samples with the homogenizer.

-tea-test-

tea-test on Nov 24 2009, 10:06 AM said:

I would say that the tissue samples from which you are isolating RNA per se are not RNase free, so the question is if those spurious amounts of RNase that might be on your extraction device will have an impact on your results. I would have more concerns about cross-contaminating your samples with the homogenizer.


ok, so cleaning or autoclaving my probe, each time, i handled a sample?

greetz, Marusya

-susanna-

gogreen on Nov 24 2009, 08:05 AM said:

Hi susanna, You can autoclave the metallic pestle and wipe all the surfaces coming contact to the tissue with RNaseZap. Don't worry If you dont have RNaseZap in the lab, Just make sure you homogenize the tissue soon and always keep it cold and add the lysis buffer ASAP after grinding. This should do the job!



I just called sigma, for his RNaseZap, but i will take while. Can i use ethanol or a detergent instead of the RNaseZAP?

greetz

-susanna-

If I were you, I would autoclave all the stuff if possible, wipe everything clean with 70% ethanol, keep the samples cold during the entire process and add the Lysis buffers (B-mercaptoethanol added if using guanidium based buffers)/ phenol:chcl3 or whatever as soon as the grinding is done

-gogreen-

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.com/techlib/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. Donít 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.

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-TJG-
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