RNA extraction expertise needed! - (Jun/29/2010 )

Hello, I've been doing RNA extractions for a while now and everythin was going fine, getting good yields and good 260/280 ratio (1.9-2.1). I recently used the bioanlayser to check the quality of my RNA and it was rubbish! very degraded with a RIN of aroud 4/5 (i here 8 what to aim for).

Anyone have any ideas what i could do to improve this?

I use RNase Zap on all surfaces and pipettes, and all plastics/ reagents used are made with RNase free stuff.

I do a pretty standard extraction from cells using TRIzol (1ml per well in 6-well plate).
1) Remove media
2) Add trizol and homogenize by pipetting
3) Transfer to sterile eppendorf and incubate at RT for 5 mins
4) Add 200 µl Chloroform
5) Vortex and incubate at room temp for 5 min
6) Centrifuge at 12, 000 rpm for 15 min at room temp.
7) Recover upper phase to fresh 1.5ml eppendorf
8) Save and freeze (-20ºC) protein and DNA phases
9) Add 6.25ull 4 mg/ml glycogen
10) Add 500 µl Isopropanol
11) Add 250 µl high salt solution (1.2M NaCl, 0.8M Na-Citrate)
12) Incubate at -20ºC for 1 hour
13) Centrifuge at 12,000 rpm for 30 min at 4ºC. Discard supernatant.
14) Wash pellet with 1 ml 75% Ethanol, vortex.
15) Centrifuge at 11,000 rpm for 5 min at 4ºC. Discard supernatant.
16) Resuspend in 35 µl RNase free water and votex`. Store at -80ºC

Everything is done very carefully, water/salt solution is aliquoted by pouring into sterile eppendorfs to avoid touching. Everything is performed on ice from step 6.

Please help!!

Hi,
correct me if I am wrong, but you recveived good results until recently and now, with the same set of reagents, it's just crap? Did you check before with the BioAnalyzer?

If that happens, first of all exchange all reagents. You may also check every reagents step by step if it is contaminated, but thats quite a workload.

Good way to list all steps carefully from the beginning on - much easier to follow instead of just reading: Degraded RNA - what to do?

I am a little confused too -- have you had good bioanalyzer results with these cells in the past?

To determine whether you have RNAse contamination, split one of your RNA samples and put half at -80 and half at room temperature overnight. Compare them on the bioanalyzer the next day. If the samples show different peaks, you have an RNase problem; otherwise, the electropherograms should be identical.

Other suggestions:

Try steps 1 and 2 on ice with cold TRIzol.

On step 5, shake by hand 15-30s instead of vortexing and try to be gentle when homogenizing in step 2 (it is possible that you are being too rough and shearing the RNA).

In my experience, Ambion's RNaqueous kit usually produces high quality RNA; if you haven't had good bioanalyzer results in the past, you may want to try that kit to get a baseline of what you can achieve -- even with the same protocols, some samples give better RIN numbers than others.