COMPLETE RNA degradation - (Aug/16/2010 )

NemomeN007 on Thu Aug 19 01:44:31 2010 said:

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.

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.

NemomeN007 on Thu Aug 19 13:49:44 2010 said:

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.

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.

NemomeN007 on Thu Aug 19 14:51:12 2010 said:

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.

Yes..DEPC-water or I usually buy ultrapure water from Invitrogen...and the pellet goes back into solution fine as long as it's not over dried. As far as OD is concerned, it's always worked fine...with ratios from 1.6 to 1.8, and for OD readings, (I will dilute into TE and blank with TE as pure water gives inconsistent readings). Usually not concerned since I'm doing a Northern or RT, so it gets internally controlled.

An update:

After changing everything, I tried to isolate some fresh samples. It turns out that it was my sample buffer--there must have been something in it that was degrading the RNA. After making fresh sample buffer, I saw three very pretty RNA bands in my sample on the agarose gel, plus OD readings of 1.7. Additionally, I took some older RNA and was able to see bands.

Thanks all for your advice. Everyone provided very useful comments!

-b

Ah. I thought that you were extracting directly from the dish using trizol...

NemomeN007 on Fri Aug 20 15:08:28 2010 said:

I usually do this. I used to trypsinize and spin down to pellet, but I found that this would decrease my yield.