RNA degredation - Extracting RNA from Brain Tissue (Feb/08/2006 )
Hi everyone, this is my first post here.
I have a question. I have read through the archives too - but I don't know if they specifically answer my question.
I have been trying to extract good quality RNA out of Brain tissue. I'm using Trizol and making sure I keep everything on ice. I clean down the area, use RNAse/DNAse free barrier tips.
The only thing is that last time I got so much RNA pellet - that it was a trouble to resuspend. So I left it on the bench in DEPC treated water to 'redissolve' - It was for more than an hour. Could this have lead to degredation?
I ran DNase treated RNA on a Formaldehyde gel and there is just a lovely streak and no bands at 16S and 28S.
Any suggestions as to a point where the degredation may have occured?
1h RT may be the critical point...
Are you sure of your DEPC treated water ? (first use, fresh, etc...)
Clean your work area with DEPC treated water and keep it on ice while dissolving your pellet and if your pellet is of big size then reduce tissue volume. and slowly not vigorously dissole RNA pellet with pipetting, One hr is too much time. it will degrade in less than 30 minutes, Autoclave ÿour DEPC water and keep the tips in DEPC treated water for whole night . Oven backed your all glassware in which you keep your solution like ethanol and isoproponal. Just wash it and autoclave but some moisture remains in it so oven bake all the glassware for 48 hrs unless it becomes completely dry. Also follow some bench clean up protocols.
Make sure you treat your gel box and casting apparatus so there is no RNAses there also. I have forgotten to do this and get degraded RNA. Clean the box and casting tray and rerun and see intact RNA.
Thankyou very much for your input everyone!
About the tips - soaking them?? I don't understand that point - I'm using RNase/DNase free tips.
The DEPC Treated water was not first use - it's the Ambion bought stuff - only used by one other person.
I cleaned out the gel apparatus with Pyroneg, RO then MilliQ H20.
Does the presence of DEPC eliminate RNases?
When letting the ethanol evaporate - should I still keep the tubes on ice?
I will go with less brain next time!!
Thanks for your help - I'll try your suggestions. If anyone has anymore - please, feel free to mention them. The more the better!!
Yes every thing should be DEPC treated. RNA is too delicate even your breath currents can degrade it . I got lot of trouble when i was isolating RNA from Cotton fiber just fiber cell. I have to make cDNA library and doing all things i am not able to get that so i used tips which is DEPC treated after this I am able to get RNA of good quaility. May be this is because of handling tips in a very casual way but you can skip this step if you are sure about your tips. I am pasting some text as such. Good Luck for your RNA.
"Tips & Tubes
Tips and tubes can be an easily overlooked source of RNase contamination. Merely autoclaving will not destroy all RNase activity, since these enzymes are very robust and can regain partial activity upon cooling to room temperature. Always use tips and tubes that have been tested and certified RNase-free. "
file as attached as well
In the past week, all of my freshly isolated RNA has turned out degraded. In the past 5-6 months, I have never had this problem...
After the first set this week was all completely degraded, I replaced all working solutions of buffers and water from the stock solutions that have never been opened on open bench space. But most of my 2nd set of samples were all degraded too!
I am using RNase-free tubes and tips, but these are kept on open bench space. How many people use hoods for their RNA/PCR preps? Would you recommend me tossing all of my current boxes of tips?
I use TRIzol reagent, and I was wondering if RNases can survive in stock solutions of chloroform, isopropanol, or 100% EtOH?? I was told that alcohol inhibits RNase activity - is that true? Sorry if these are naive questions - just my reminder, I guess, that a lot of this is still new to me!
In my old lab we used a hood for RNA work always. Not in this lab though
RNase will not survive in the chemicals you mentioned. I would suggest making up fresh buffers (in particular whatever you use for elution) and using freshly opened tips and tubes. Also I would suggest decontaminating your bench space and pipettes with RNaseZAP, and also giving your homogeniser (if you use one) a good clean with ZAP as well
That shoudl rule out any expgenous RNAse factors. If it is still coming out degraded after that then the problem lies somewhere else (we can deal with that if it happens)
Thanks, John! It helps a lot to know that my chemicals are ok. If the problem is not solved by doing a thorough cleaning and new solutions, I'll definitely be back here.