Problem in RNA isolation, degraded, even though I pay extra attention to prevent - (Jan/30/2006 )
I was trying to isolate RNA from mouse tissue to do microarray study, I have tried Trizol/Trizol LS, Qiagen Mini column, and Qiagen midi column. However, all I can found out is I can see clear nice 28S, 15S on the gel and once I send to have the aligement test, the core people said my RNA was degraded. I am pretty sure that I am really aware of gloves, use RNase away to wipe all the pipitte, DEPC water etc. And I could not think of any way I can do to solve problem. Plus whether harshly pipetting will cause RNA fragmentation? I just know Genomic DNA should not be done by that and have no idea whether RNA will have the same issue even if RNA is pretty small.
Do you check the OD at each step (i.e. after extraction, after DNase treatment, after reverse transcriptase, etc.)?
You need very high yields of RNA/cDNA in order for microarrays to work. You might want to consider amplifying RNA.
I'd consider 3 ug the bare minimum (at least for affymetrix).
I did, the yield is good, A260/A280 ratio is always above 1.8, below 2.1, concentrationis around 2.5 ug/ul and the total volumn is around 25 ul. my problem is I just don't what I could improve, seems I tried everything I could but still got degraded RNA.
Are you getting that OD after DNase treatment?
Exactly what protocol are you using?
Can you check the RIN using a bioanalyser?
you can work in cold room that prevents the degradation...
Sorry guys reply such late.
I used different protocal, 1) I used Trizol isolation RNA first and then use TRI zol LS purification again.
2) I used the Qiagen RNeasy mini column tried purified it.
I tried in the cold room, and still degradation according to the bioanalyzer.
"Can you check the RIN using a bioanalyser?" what is that? I am not the one doing this, I am not sure how to check that. Also whether by just pipeting will cause RNA fragmentation? Since RNA is pretty small, I would not think so, but maybe I don't know.
pipetting will not that degrade RNA. Assuming it's not a very intensive pipetting up and down that's ok. facing degradation, i've noticed a guideline for RNA extraction : the quicker the better. Reducing the time for the manip drive a good reduction of degradation of my RNA's.
how do you store them? (in DEPC water? formamide?...) is there o quite long time before you prepare tissues and start the extraction?