Need help w/ RNA Isolation from you lab pros??

[Lux Aeterna]

Hey everyone,

Since all of you MSTPers are super well-versed in lab and lab techniques, I hope you don't mind me venturing in from pre-allo and asking you for some tips on RNA isolation. I'm sure it is something many of you have done a million times, but I'm doing it for my first time and I'm having some issues.

Specifically, I am talking about RNA extraction from tissue (mouse lung). We harvested the mouse lung and snap froze it in smaller pieces. I pulverized the lung while frozen and sonnicated/homogenized it with RNA Stat-60 and proceeded with the isolation. When I sent it up to the Core facility to check the 28S: 18S ratio with the agilent bioanalyzer, the RNA was all degraded.

OK, so I thought, maybe its cuz I'm doing it for the first time and my technique is contamination-prone (which I didn't think it was). I did it again with another person in lab who is more well versed with RNA isolation (usually from cells though), and the RNA was still degraded. I was super vigilant in making sure I wasn't contamination-prone. But still... degraded RNA. (Oh yeah, concentrations look ok, as does the 260/280 nm ratio).

what do you guys suggest? a Qiagen prep kit? use a glass homogenizer instead of the sonnication probe? Am I overlooking some key part? Am I just not getting it?

I really want to get this right so I can move on to the cDNA and real time PCR part of the project. I feel stuck on this step, and I don't know what my next move should be in attempting this isolation.

Thanks for reading this post, sorry it was so long. I feel like this is something that is fairly easy for people, but I'm the loser who can't get it right!

Lux

 

[surge]

Without knowing much detail, I've heard of people having this problem with live tissue isolation.

You are, of course, using sterile technique, a tissue culture hood, etc., right?
Have you thought about using RNAse inhibitors?
(such as Ambion's http://www.ambion.com/catalog/ProdGrps.html?fkApp=12&fkSubApp=38 ?)

 

[BandGeek]

I would recommend the Tri-Reagent procedure from Molecular Research Centers. http://www.mrcgene.com/tri.htm
I have used this procedure for over four years and have never had problems.
I have a few tips as well.
First of all, ALWAYS have a dilution tube. That way you can go back to the original sample. Agilent doesn't require much RNA, so you can easily make a dilution up for Agilent and FREEZE immediately your stock. If your RNA dilution is degraded, then make another dilution.
Also, I would recommend using some RNAse zap on your sonicator after you throughly clean it.

I hope this helps you out.

 

[Lux Aeterna]

I just tried to post reply and it didn't go through. Blah.

Surge--Yep, sterile technique, hood, etc. I will look into that link you provided.

BandGeek--I have been using RNase Zap /Away on the sonnicator, etc. I also am making dilutions for the agilent, since I think you can get away with just giving them 100 ng of RNA.
I will look into the procedure you use through the MRC link you provided.

Thank you both for your responses!

 

[Aptamer]

Yes! Use Qiagen prep kits. If Qiagen makes it, you use it. They work oh so beautifully.

Definitely, when you work with RNA, make sure everything you use is RNase free. To go along with the Ambion suggestion, I recommend using Ambion's RNaseZap wipes and wipe down your bench and pipetman. Ambion also has a RNase test kit, RNaseAlert. The assay takes thirty minutes to do and you can eyeball to see if your sample has RNase or not.

What kind of water are you using? Is it Milli-Q water? Are you making sure it is completely sterile by autoclaving or filtration? You can purchase DEPC-treated water and/or nuclease-free water to use, depending on protocol.

Are your tips and tubes certified DNase/pyrogen/RNase-free? Do you autoclave the tubes? Are your tips aerosol-resistant, to reduce sample carryover? Do you have a positive-displacement pipetman that you can use?

Sorry for all the questions. Hopefully it helps (more likely annoy).

 

[Treg]

Two words: Qiagen RNEasy

Great for tissue extractions and any other source you might have. I have prepared samples for Taqman RT-PCR, and my labmate has done tons of RNAse protection assays using RNA made from the kit. It is great because you can homogenize fresh tissue in Buffer RLT and freeze it at -70 C to use up to 6 months later. To be really careful about your homogenization, I would suggest purchasing disposable eppendorf tubes that come with disposable pestles that fit perfectly (both are certified RNAse/DNAse free). If you want the source PM me and I will get it at the lab tomorrow.

Seriously-qiagen is $$ well spent.

Also, if $$ is a factor, it is really easy to make DEPC water. Again, PM me if you want a protocol.

Good luck!

Treg

 

[ckent]

Well, I've never had to isolate RNA myself, but I do recall using a lot of qiagen products. They are really great, they can be a bit expensive, but they make life soo much easier. Also, I agree with many of the reccomendations being posted here. You should realize that your skin has RNAase on it, so anything you touch, you should consider contaminated. RNA is a huge pain to isolate, it degrades fairly quickly after cells or animals die, so you might also consider seeing if there is any way you can speed up your procedure time as well.

 

[vixenell]

I second (or third)the Qiagen suggestion... I used it this summer, and never had a problem with isolation.

Another thing... make sure that everything is on ice at any moment when you're not working with it. Don't stick the tubes all the way into the ice, as it is not clean. Make sure that all of your reagents are being stored at the proper temp. Seems simple, but it can be easy to forget.

Also, even if you are wearing gloves, avoid touching the insides of the eppendorf tubes; gloves may protect you, but they are not clean and can contaminate your data.

Perhaps you're doing too many samples at once? If thats the case, try running through the protocol with about three samples, or 'extra' tissue to make sure that you can do the protocol without wasting needed tissue. Once you get that to work, you know that the problem does not lie with you.

 

[hockebob]

it might also be good to do it with another source of RNA entirely as a positive control (ie- cells grown in culture, something fresh that hasn't be frozen at -80, etc). that way, if both fail you know it's something wrong with your technique, but if only the tissue RNA fails then perhaps something happened during the tissue isolation and freeze process. by the way, you said that you snap froze the tissue right? what does that entail? the reason i ask is that people in my lab often use OCT compound to freeze live tissue for in situ hybridization and it's usually very successful.

aaron

 

[Lux Aeterna]

hey guys,

thank you all for the great responses.

next week in lab I am gonna attempt the isolation with the Qiagen kit:: the RNeasy... *hopefully this will work!* I think Qiagen has a quick procedure time which will benefit the RNA extraction.



I've been using DEPC water. Everything is autoclaved, eppendorfs are certified RNase/DNase-free. Been using RNA Zap as well. I'm gloved up always. I will check to see how I've been putting my samples on ice. They are always on ice, and I don't think I stick them way down in the ice bucket, but you know, it is something I never really gave much thought too--which could be a bad thing! lol. Snap froze via immersion in liquid nitrogen.

For you Qiagen users, any helpful tips you have for the protocol or is it pretty straight forward with that pink column and eluting the various reagents ?

thanks!!

 

[Gut Shot]

There is a wonderful device made by Biospec: the mini bead-beater. I routinely used one to extract high-quality RNA from fungi with great results (I used Qiagen's RNeasy kit, as well).

Basically you snap-freeze the tissue in liquid nitrogen in a 2 ml screw cap tube containing glass beads. Then add the requisite amount of the first RNeasy reagent to the frozen sample, pop it in to the bead-beater, and it does the rest. The RNA is freed from the cells rapidly and directly into the RNA extraction solution. After spinning out the debris it is a simple matter to follow the rest of the Qiagen protocol.

Getting a bead beater is a relatively small investment, and the things are quite versatile. It might be a good investment for your lab.

Good luck.

 

[Fixed Gear]

First off, you need to do a positive control to make sure that the RNA in the mouse lung isn't toast. You could be doing a great job extracting RNA that will always look like poop.

Second, for skeletal muscle samples and ES cells, I have used TRIzol (from Invitrogen) with tremendous success. Tissue is placed into an aliquote of TRIzol and then homogenized. The TRIzol then helps to protect the RNAs.

Lastly, youdon't need to do RNA extraction in a hood. That's overkill. A clean bench and pipetors is sufficient. In our lab we have a PCR/RNA bench and we all get good RNA.