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Handling RNA - How to avoid RNase contamination


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17 replies to this topic

#1 susanna

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Posted 24 November 2009 - 03:15 AM

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

#2 gogreen

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Posted 24 November 2009 - 07:05 AM

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!

#3 susanna

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Posted 24 November 2009 - 08:03 AM

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

#4 mdfenko

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Posted 24 November 2009 - 08:19 AM

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.
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#5 gogreen

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Posted 24 November 2009 - 09:41 AM

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

#6 tea-test

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Posted 24 November 2009 - 10:06 AM

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.
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#7 susanna

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Posted 25 November 2009 - 12:34 AM

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

#8 susanna

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Posted 25 November 2009 - 12:56 AM

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

#9 gogreen

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Posted 25 November 2009 - 01:22 AM

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

#10 TJG

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Posted 08 December 2009 - 02:31 PM

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. 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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#11 kip

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Posted 22 December 2009 - 08:03 PM

question- how does one go about posting a question??

#12 William Parkar

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Posted 21 April 2010 - 10:17 PM

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!


http://www.flashpapers.com

#13 apbrown

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Posted 01 July 2010 - 06:59 AM

i HAVE A QUESTION REGARDING RNA EXTRACTION USING TRIZOL REAGENT. WHY DO I HEAT THE TRIZOL? IS IT GOOD TO HEAT IT OR DOES IT MATTER?

#14 DNAgeek

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Posted 03 September 2010 - 06:47 PM

Be concerned with RNases in the tissue - work quickly, on ice if possible.

If you are using a high-salt buffer to homogenize the tissue in, that should inhibit RNases somewhat.
I homogenize my tissue already in Trizol, which denatures all proteins, including RNases.

To decontaminate, you can use 0.5% SDS and then wipe everything down with 70% ethanol made with DEPC'd H2O.
Most importantly, I clean everything I touch, including my gloves, with 0.5% SDS and wipe with 70% EtOH to prevent any RNase contamination.

I don't autoclave, it's harsh on the equipment and not 100% effective on RNases.

#15 Dodger

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Posted 22 November 2010 - 04:20 PM

It might be a little late to post an answer to this thread due to it's age, but since it's pinned...

We usually prepare 4x 50 mL tubes. Fill three with 40 mL DEPC treated water, and one with 100% ethanol.
One of the DEPC treated water tubes gets 5-10 squirts of RNase Zap.
Between each sample, the homogenizer is dipped into 100% ethanol, RNase Zap water, and 2x DEPC treated water in turn.
Each time turning the homogenizer on for about 15-30s.

This is fairly quick and seems to work well for us.




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