19 replies to this topic

[Ahrenhase]

I'm having a little problem here with the TRIzol method.  Everything is pretty clear cut until you get to the RNA wash.  After you precipitate the RNA in isopropanol, you give it a wash with 75% ethanol in DEPC water.  I mixed the sample by vortexing then spun it down.  After centrifugation, I could definately see a pellet at the bottom of the tube.  Now this is the part I don't understand.  The very next step after spinning it down is to briefly let the pellet air dry.  I assumed they mean after decanting most of the ethanol off (because there's 1mL of it).  So that's what I did.  It also says not to overdry pellet.  So what I did was took a 20ul pippette and drew most of the ethanol off on the side of the tube opposing the pellet.  I got this down to say 10ul of ethanol left but I could still see the pellet swimming around in the ethanol.  I figured I'd try to get most of it out because I don't want a REALLY low 260/230.  So I stuck a clean 20 ul pippette tip into the bottom of the tube to break the surface tension of the ethanol, and it worked.  I got another 5 ul out.  There was still about 4-6ul ethanol left so I left it air dry for about3 min.  By this time I could still see some ethanol but I didn't want to overdy the pellet, so I redissolved it in 20 ul RNase free water and ran it on the nanodrop.  I got a really low 260/230 of about .5 and my 260/280 was 1.5.  What happened?

Edited by Ahrenhase, 22 May 2009 - 04:50 AM.

[Velella]

I think that discarding all the ethanol off your RNA pellet is one oh the most challenging steps of Trizol RNA extraction.  After spinning the pellet down in ethanol, I usually prefer to turn the tube once upside down (very delicately) on a blotting paper, only once, then it's very important to keep the tube always upside down, try to aspirate with the void and a tip some residual drops on the walls of the tube (of course being very careful not to aspirate the pellet) and then put the tube always upside down on the paper (don't put the tube right again because the residual drop of EtOH would go on the pellet and detach it from the plastic) for 1 or 2 minutes, than I put it into the bell with the void (sorry, I really don't know the name in english) to let it air dry for at least 5 minutes. Then Icheck the pellet, if I don't see residual EtOH I resuspend the pellet, otherwise I put the tube again in the bell for 2 minutes, not more. With this protocol usually the pellet is dry, but naybe it depends on the dimension of your pellet. hen it's dry it becomes transparent.
When I resuspend the pellet I simply put the RNase free water and try to detouch the pellet pipetting up and down (usually it becomes filamentous), when the pellet is detouched from the plastic I let the pellet solubilize for al least 3-4 hours at 4°C. Then I check the tubes, if I see the filamentous pellet yet, I pipette again up and down till I don't see it anymore.
I hope this will be helpful!!!

[Trof]

The protocol says don't overdry but that doesn't mean you should keep some ethanol in the tube. At the final step I drew all the ethanol with a pipette, and then let the pelet which is still always wet to dry. The pelet should be stil visible, i.e. whitish, it shouldn't turn to transparent.
Anyway AFAIK the overdrying only makes troubles with dissolving the pelet, so I air dry for 10 minutes and then add warm (65 deg) DEPC water an let dissolve 10 minutes on heatblock on 65.

[lsek]

Hi.
I've got the same problem. Finally decided to do extra wash with 70 to 75% EtOH. NOTE that each EtOH wash should be done by disrupting the pellet and pipetting it up and down (don't worry about losing your RNA). If the problem persist, dissolve RNA in DEPC water, ethanol precipitate again (i.e. 1/10 vol 3M NaOAc and 2.5 vol EtOH) and you'll get a much cleaner OD read. But please make sure you take precaution to avoid RNA degradation.

Well, it seems that the low a260/a230 is due to guanidinium carryover (or phenol etc).

[Ahrenhase]

lsek, on May 22 2009, 10:12 AM, said:

Hi.
I've got the same problem. Finally decided to do extra wash with 70 to 75% EtOH. NOTE that each EtOH wash should be done by disrupting the pellet and pipetting it up and down (don't worry about losing your RNA). If the problem persist, dissolve RNA in DEPC water, ethanol precipitate again (i.e. 1/10 vol 3M NaOAc and 2.5 vol EtOH) and you'll get a much cleaner OD read. But please make sure you take precaution to avoid RNA degradation.

Well, it seems that the low a260/a230 is due to guanidinium carryover (or phenol etc).

Low 260/230 are also indicative of ethanol carryover too,  i think

[Telomerase]

If it's tissue isolation, or otherwise unfavorable conditions, and the RNA is scarce, I wouldn't recommend drying by heating. Just spinning it short after the last wash, and removing excess ethanol, then drying in RT is enough. I wait until the pellet turns transparent, but do not leave it waiting afterwards. You just have to check often. If it's not heated, there's no big risk of overdrying. Otherwise you might get a gummy pellet, no fun.
And yes, the low 260/230 is more often residue ethanol.

Edited by Telomerase, 22 May 2009 - 08:57 AM.

[Ahrenhase]

Great, thanks everyone.  I apparently didn't give the ethanol enough time to evaporate.  Next time I"m going to do a final wash with 95% so it dries faster.

[Ahrenhase]

I tried it again, this time adding a 95% ethanol wash at the end.  I clearly saw a pellet at the bottom of the tube.  I let the remaining 2ul of 95% ethanol (probably mostly DEPC water) evaporate off the pellet until it was semi-opaque.  I could clearly see the pellet and there was a little white halo around it.  So I added 30ul Gibco RNase DNase free water and sat it on ice for about 30 min. before running a nanodrop.  I got nothing.  So I did as the protocol says and heated the sample up to 55C for 10 min and reran it.  Still nothing.  I read that RNA isn't easily soluble in water but TE buffer is out of the question as my sample is for qRTPCR.  I will run it out on a gel tomorrow to see truly whether there's anything in my sample or not.  

Does anyone know of another method other than TRIzol that isn't as finicky?  I typically use Qiagen minikits, but the cells I'm using seem to be getting clogged in the QiaShredder and I never get any product.  .

[phage434]

TE is definitely NOT out of the question.  The only problem with TE is the (very small) amount of EDTA, 1 mM.  When you set up your PCR reaction, this is diluted at least 20x, so you end up with 50 uM EDTA in your PCR reaction, which will hardly affect it at all, given the 1-2 mM magnesium in a typical buffer.

The EDTA will not inactivate RNAses, so it will not have the benefits that it has in DNA storage, where DNAses are inactivated by chelation of Mg++.

If you insist on eliminating the EDTA, then use 10 mM Tris pH 7.5 to dissolve your RNA.  Water is a poor second choice.

[Velella]

I used Qiagen RNeasy minikits with many different cells and I always had appreciable results but I never used QiaShredder to homogenize, I simply used an insulin syringe (1 ml- 25GA). Briefly I usually scrape the cells with RLT buffer (but sometimes I also added RLT to a cell pellet stored at minus 80 and I had still good results even if perhaps a bit less yeld) then I collect the lysate in the tube and pass it through the syringe 5 times and then I add ethanol, load on the column and so on.
If the problem is the QiaShredder in this way you should not lose material, I used this method with primary microglia cells that drove me desperate with Trizol because they give  a really very low yeld of RNA, and I was able to gain a good RNA without losing it. I know that they say that QiaShredder should not make you waste material, but in my experience needles work well!
RNA isn't very soluble in water, this is why with Trizol I usually pipette the pellet up and down to disrupt it better before leaving it at 4 degrees for 3-4 hours...but it's odd that you don't read anything at all at nanodrop...did you check after adding the water, waiting 30 min and also heating it,  the condition of the pellet? If the pellet is not solubilized (and if this is why you don'tread anything) you usually see it, if you look at the tube against the light and pipette up and down, like a almost transparent filamentous thing fluctuating in the water. At least, this is my experience...
There is a third option I used on liver tissue (but I read that you can use it also with cells): loading the aqueous phase of Trizol extraction (after chloroform and centrifuge) after having added equal amount of ethanol 70%, on the column and then go on with the same protocol. With tissue it worked well, I don't know aith cells but you can try!
Good luck!

[Ahrenhase]

Yeah I tried the needle thing too.  I saw the filamentous noodle floating around the bottom of the tube, so I don't know where it went.  I even ran a gel and there was absolutely nothing.  I think I might try using the aqueous layer from TRIzol on the minikits.

[lsek]

Hi,

I'm not too worry about the residual ethanol in RNA pellet. Air "drying" it for about 5 to 10 min is already sufficient. "Drying" here means allowing the 70% ethanol to evaporate to a conc of below 30-50% ethanol content. However, note that water will not evaporate as easily as ethanol.

Alternatively, heat briefly in a 50 degree C oven for say 1 to 2 min. Then air dry, allowing EtOH to evaporate while residual water remain in the tube. There'll be droplets at the side of tube and if you are really particular about completely eliminating all droplets, use a clean tissue to blot out these droplets.

However, I sincerely believe that your low A260/A230 is due to guanidinium carryover. I've had the same problem and after a second ethanol precipitation, the ratio is at a very healthy level. Example, before EtOH precipitation I'll get around 0.9 to 1.5 reading, now... I'm getting >2.0.

Good luck.

P.s. there's a link for troubleshooting low a260/a230 for rna @ http://wheat.pw.usda.../pro/tb087.html. NOTE: I'm using Trizol for RNA isolation.

Edited by lsek, 28 May 2009 - 07:06 AM.

[eldon]

Sounds like you're being TOO delicate...need to work fast and without concern with RNA but in an RNAse-free zone.

Having done 1000's of RNA preps using TRIzol or RNeasy etc. I recommend not using TRIzol. RNeasy with on-column DNAse digestion is far superior and doesn't require nasty extractions with phenol. Total RNA preps using RNeasy consistently outperforms TRIzol if your RNA is for qRT-PCR or microarray, but equal in northerns.

[Clare]

eldon, on May 28 2009, 07:28 PM, said:

Sounds like you're being TOO delicate...need to work fast and without concern with RNA but in an RNAse-free zone.

Having done 1000's of RNA preps using TRIzol or RNeasy etc. I recommend not using TRIzol. RNeasy with on-column DNAse digestion is far superior and doesn't require nasty extractions with phenol. Total RNA preps using RNeasy consistently outperforms TRIzol if your RNA is for qRT-PCR or microarray, but equal in northerns.

But don't you lose all the miRNAs with the RNeasy columns?

Clare

[eldon]

Quote

But don't you lose all the miRNAs with the RNeasy columns?

Clare

No.