DendWrite

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I hope this sort of topic isn't frowned upon here (I read some topics like this a while back), but I'd like to hear from some of you guys on this, since I'm sure you've done this before.

I'm doing RNA extraction from tissue by homogenizing in Trizol (1mL), adding chloroform (200µL), vortex well, spin down, take off top, clear layer (avoiding protein/DNA layers), add 500µL 2-propanol, vortex, spin, aspirate, add 500µL 70% EtOH, vortex, spin, aspirate, resuspend in DEPC-treated H2O.

I can get an OK yield, but my problem is always PURITY (OD260/OD280)! I cannot, for the life of me, get above 1.5 or 1.6, yet I know that I want 1.9+. Do you have any suggestions as to what I might be doing wrong? I know the most important step is not aspirating any of the protein/DNA layer after the first spin. But I'm spinning at 14k RPM for 30 minutes, so it's pretty much as solid as it's going to get, and I'm using a small pipet and pipetting as SLOOOWLY as possible so as not to disturb the layers below. Also, this last time I did it, I even left about 50µL of the clear layer that I could likely have taken off, and still, purity values of 1.5 or 1.6. I know that I'm not the greatest pipetter in the world, but I've had good results with real-time PCR, etc.

I know that you can just do a purification step, but I'd like to try to avoid it. I know lots of people that can get 1.9+ without purification step. Are there any secret tips you guys have?

I don't know if Trizol goes bad either...
 

muchomaas

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Can you check to see if that difference is even relevant for your downstream application?

Can you check to see if you extract something else (like a cell line) if the 260/280 is better? Could be sample-dependent.

Worst case scenario replace all reagents and start again.

For what its worth, I've done a ton of rna extractions, and I only vortex really the trizol/chloroform mix.
 
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I had trouble isolating total RNA with good 260/280 ratio with cardiomyocytes (presumably because of the large amounts of contractile proteins, etc.) and I added a step where I did a proteinase K digest.

Here's an aritcle (they use the Qiagen RNEasy kit but add a proteinase K digestion step): http://www.clinchem.org/cgi/content/full/50/5/975

Hope this helps.
 

reine1jb

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I agree with trying it on another sample, there are samples which can be extremely hard to extract RNA from just because of the nature of the tissue.

When I used trizol, I always did the EtOH wash twice. The first couple times I did it once and had similar results as you. Once I started doing the EtOH wash 2X, i instantly had 260/280's of 1.90-2. Also, I don't know if you do this, but I did the 30min (it's been awhile but I'm pretty sure I only did it for 15) spin at 4 degrees Celsius.

Have you ever tried to run your RNA out on a gel? If so, do you see the two (at least I think it is two) rRNA bands or Is it degraded (smear), or is there a high molecular weight fragment band (could be DNA contamination)?

because your Real-time is working (assuming you're using your isolated RNA for the RT rxns), it seems to suggest that your RNA is not degraded. Do you include a control rxn in your real-times in which you put just your isolated RNA in as template (you should not get any results from this). If you do get results, then you're probably just amplifying genomic DNA. Do your primers span exon exon boundaries?
 

StIGMA

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3 ideas:

You didn't mention using a sedimentation agent (2.5-30ug glycogen per tube). You may be losing a lot of sample and not realize it. (Your resuspension may be incomplete if you are transfering tubes).

Consider using DNase. Why not incubate with DNAse after the first extaction?

Also, you did not mention how long you are incubating your sample in trizol initially and at what temperature. Extend this duration for longer than what you are doing (I think I used 30min), and do it at room temp. (Again, a yield issue).

(Note: haven't checked these protocols in about a year, and I worked with cell culture, not tissue).
 
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DendWrite

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Wow...thanks so much for all of the helpful (and quick) feedback.

muchomaas:
As far as why I want the higher quality: I'm going to do a microarray assay which, as far as I've read, purity is of crucial importance (also, my PI will get really mad if we have to repeat that kind of experiment ... $$). I've tried it with cells and tissue and gotten similar results.

reine1jb:
In the past I've used isolated RNA with 1.5 to 1.6 purity for real-time PCR and it worked fine. , I run the control as you mentioned, and there's only amplification of my cDNA...not the RNA. As you mentioned, I do all the spins at in the 4˚C coldroom. I will definitely try the second EtOH wash next time. Yes, the primers I'm using span exon-exon boundaries.

alphacentauri1:
This looks like it will be very helpful. I'm working with cardiomyocytes as well. Thanks a lot for the link!

StIGMA:
I've done a purification step previously using DNAase, but it didn't really improve my purity, and I didn't notice a significant difference when I did the real-time PCR (which was all I was using the RNA for at the time). I generally incubate with Trizol for about 10 minutes at room temperature. I'll try the longer incubation next time.

So...for next time, I'll do the longer incubation and second EtOH wash. I'm getting acceptable yields for what I'm doing with it, so right now quality's my biggest concern. When I get that up, I'll try the sedimentation agent as well to see if I can enhance the yield. If I'm still having purity issues, I'll try proteinase K / DNase.

I'll post back when I've figured out what works best (hopefully this will occur sooner rather than later :D). Thanks again for all the help.
 

muchomaas

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See if your institution has a bioanalyzer available. This seems to be the standard for RNA quality for microarray/sequencing applications now. I don't know if anyone every put that much stock in the OD ratios . . .
 

CielloStelatto

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A couple of thoughts:

I agree with muchomaas that a bioanalyzer might be a good idea if it's readily available - otherwise it might only be necessary as a later step to test RNA quality. Although the OD ratio's aren't perfect and don't give a full picture, it's definitely a starting point, and a very low ratio for all of your samples (from different lines or tissues) is probably indicative of some other issue.

Someone previously mentioned this, but have you run your RNA on a gel? and what does it look like?

Also, make sure that you are using an amount of tissue that the TRIzol can handle (I think 10% of the reagent volume). Any more than that and the RNA won't all be stabilized. I once tried using more culture cells than recommended with TRI reagent, and it wasn't clean at all.

You mention that you're not sure if TRIzol goes bad. If you're suggesting that your bottle has expired, then get yourself a new one.

I don't fully remember the protocol for TRIzol, but for both RNAzol and TRI reagent, the chloroform/BCP step was hand mixed and not vortexed - perhaps that would make a different for trizol as well?
 

Drexon

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I've had trouble getting high enough 260/280 before. what kind of lysis agent are you using .. perhaps try a different lysis reagent ???

perhaps buy a kit from Qiagen or macherey nagel and see if you get the same results? just one for the sake of having a lab common kit for people to use to verify results.
 

FSAP

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I always get about 1.5 when I isolate RNA from lung homogenates. FWIW, we sent our samples to the facility at JHU and they did both the extraction and microarray for us. Their OD ratio was around 1.5 on average as well and it seemed to work out fine.
 

lazy

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All you need is enough for your RT, not the biggest yield you could possibly get. Think about that when you do the first aspiration (the one after the trizol chloroform spin).

It really isn't a big deal if you dont get as much as you can from above that layer, as long as you get enough to do your experiment, so don't fret about wasting some unless you only have a very limited supply of cells to start with.

What are you extracting the RNA from? Perhaps you need to homogenize it a lot better before the first spin?