18 replies to this topic

[btc8]

Hello,

I've isolated RNA several times in lab, and we use the TRIzol method.  I've had no troubles with it in the past, but the past few weeks I've experienced major problems in the area of degradation.  Usually after the ethanol wash we resuspend in DEPC-H2O then we run a 1% agarose gel to verify the integrity of the RNA.  No matter how careful I've been, I NEVER see any sign of a band in my samples.  No smears; no faint bands; just no bands.  It's very disheartening considering I've had no problems in the past doing RNA, I've changed every single solution, gotten new tips, tubes, etc., and my 260/280 readings for these degraded samples have all been on average around 1.8-1.9.

Does anyone have any advice or experience with something like this?  I'm still isolating from the same cell lines I have in the past.

Thank you for your help.

[perneseblue]

Do you use filter tips? Might the contaminant be in the pipette barrel?

What about the alcohol? Or any other reagent?

[phage434]

Could be RNAse in your agarose gels or running buffer.  Or you gel trays, combs, loading dye...

[btc8]

perneseblue, on 16 August 2010 - 05:49 PM, said:

Do you use filter tips? Might the contaminant be in the pipette barrel?

What about the alcohol? Or any other reagent?

I was thinking about using filter tips; I'm going to isolate Wednesday and try that.

I usually wipe down my bench and pipettes and whatnot first with 70 % EtOH, then with RNAZAP, then I rinse with ddH2O.

phage434, on 16 August 2010 - 06:00 PM, said:

Could be RNAse in your agarose gels or running buffer.  Or you gel trays, combs, loading dye...

I thought about this after the first time I experienced the degradation. But I rinse them thoroughly with ddH2O, then RNAZAP, then again with ddH2O.

I was also thinking it might be an incomplete lysis of samples problem--that is, there's not a lot of RNA to begin with--though this doesn't explain my OD readings. For monolayer cells, I usually just add the Trizol directly to the plate, incubate for 5 minutes, then pipette the lysate several times.  Is it necessary to scrape to cells?

Thanks for all of your suggestions.

EDIT: The loading dye is a good point.  Come to think of it, the loading dye is the only thing I did NOT change when I replaced all of my reagents.

Edited by btc8, 16 August 2010 - 07:14 PM.

[HomeBrew]

If the problem was due to RNAse contamination of a reagent or piece of equipment, wouldn't you still expect to see degradation products?  Isn't it unlikely that all the RNA would degrade completely to single nucleotides from trace contamination by an RNAse?  Does your MW ladder light up?  Do you have a "known good" sample of RNA you can include on the gel as a control?  Are you doing a DNAse digestion step?

[btc8]

HomeBrew, on 17 August 2010 - 01:08 AM, said:

If the problem was due to RNAse contamination of a reagent or piece of equipment, wouldn't you still expect to see degradation products?  Isn't it unlikely that all the RNA would degrade completely to single nucleotides from trace contamination by an RNAse?  Does your MW ladder light up?  Do you have a "known good" sample of RNA you can include on the gel as a control?  Are you doing a DNAse digestion step?

This is a good point, though I have no idea why I'm not seeing any smears.  I'm pretty much at my wits end thinking about it.  We usually don't run a MW ladder for RNA--just a quick identification of 28 and 18 to see if they're there and the RNA is legit.  Yes, we do DNAse treat the samples--running a gel before and after. I did run a ladder with one gel before because I suspected that perhaps there was something about my gel that was the problem, but surely, the ladder lit right up.  So maybe it's a problem with insufficient lysis of cells?  Still, doesn't explain my OD readings.

I have absolutely no idea, and it's doing my head in.

Thanks for your insight and advice.

[ElHo]

Maybe you can try to make cDNA and expression analyses for a housekeeping gene for 1-2 samples to see whether you really have no/degraded RNA in your samples or if the problem lies with the gel electrophoresis as your specrophotometry indicates good quality RNA.

[HomeBrew]

OD readings do not equal RNA...  Anything (including single nucleotides, organic solvents, etc.) that absorb light at the wavelength used will contribute to the OD reading.  The OD reading is telling you the degree to which your sample is optically dense; it says nothing about what's causing that density...

[btc8]

HomeBrew, on 17 August 2010 - 12:03 PM, said:

OD readings do not equal RNA...  Anything (including single nucleotides, organic solvents, etc.) that absorb light at the wavelength used will contribute to the OD reading.  The OD reading is telling you the degree to which your sample is optically dense; it says nothing about what's causing that density...

This is true.  I'm thinking it's my pipettes, so I'm going to use a different set along with barrier tips.

[NemomeN007]

What volumes are you using...and do you see "hazy cloudiness" when you EtOH precipitate after the chlor extraction?

[btc8]

NemomeN007, on 18 August 2010 - 05:44 PM, said:

What volumes are you using...and do you see "hazy cloudiness" when you EtOH precipitate after the chlor extraction?

I generally harvest from 6-well dishes with 1 ml Trizol/well.  So everything is from 1 ml onward per sample.  I don't see what you describe as anything hazy during the EtOH washes.

I suspected that my sample buffer was causing my degradation (as I changed everything except that), so I remade some and ran some old RNA that I isolated previously on an agarose gel, and lo and behold, I saw perfect 28 and 18S bands.  I was relieved when I figured this out, so I isolated some more RNA, but I didn't see any pellet at the EtOH wash, and there was nothing in the gel when I ran it.

[NemomeN007]

What I meant is that during extraction from a 6-well plate, using 1ml of trizol...after the addition of Chloroform and during the isopropanol precip. you should be able to see stuff precipitate...sort of a slight haze in your solution...if you're no seeing that, then either your trizol is no good anymore or the chloroform is bad....in either case, RNA is not being extracted.

[btc8]

NemomeN007, on 19 August 2010 - 05:49 AM, said:

What I meant is that during extraction from a 6-well plate, using 1ml of trizol...after the addition of Chloroform and during the isopropanol precip. you should be able to see stuff precipitate...sort of a slight haze in your solution...if you're no seeing that, then either your trizol is no good anymore or the chloroform is bad....in either case, RNA is not being extracted.

Gotcha.  Yes, after isopropanol addition, I invert the tubes a few times, and I can see what appears to be some tiny little crystals and hazy things moving around.

[NemomeN007]

hmmm...sounds like you're losing your pellet during the EtOH washes...are you using new microfuge tubes by chance or siliconized tubes?  Pellets have a tendency to float off during washes.
Personally, I don't do the EtOH washes and go straight to the next step with no problems.

[btc8]

NemomeN007, on 19 August 2010 - 06:51 AM, said:

hmmm...sounds like you're losing your pellet during the EtOH washes...are you using new microfuge tubes by chance or siliconized tubes?  Pellets have a tendency to float off during washes.
Personally, I don't do the EtOH washes and go straight to the next step with no problems.

Interesting.  So you go right to dissolving the pellet in DEPC water?  Does this not affect your OD or integrity?

Thanks for your suggestion.  I will try that on one sample, but leave the rest for washing because I always find that the pellet has troubles 'returning' after I wash it in EtOH.