Total RNA extraction using TRizol for isolating miRNA - (Mar/14/2007 )

Hi!
I'm fresh in this field!I want to isolate miRNA from a kind of insect. But I met a problem when I extracted total RNA using TRizol(Invitrogen).The 2:1 ratio(28S:18S) is a good indication that the RNA is completely intact. But the RNA I extrcted can not show that ratio. I repeated the experiment for many times, I still can not get the total RNA having the correct bands.I don't know why! And how should I do! Your help is very appreciated!The attachment is my electrophoresis of the total RNA.

-Garden-

what is your method ? strickly the one described by protocol ?
did you try to increase time of incubation (10') and do it on ice?
suspect your RNA is quite degraded.
Speed, lysis efficiency are the major critical points

-fred_33-

QUOTE (fred_33 @ Mar 15 2007, 11:40 PM)

Thanks for your instuction! I extracted once more yesterday.Could you think the total RNA is degraded? The second picture is 6% PAGE.

-Garden-

QUOTE (Garden @ Mar 17 2007, 11:09 AM)

Thanks for your instuction! I extracted once more yesterday.Could you think the total RNA is degraded? The second picture is 6% PAGE.

You made PAGE for RNA analysis?

I dont know if this could help but we had in our lab TRIreagent from 2 companies Sigma-Aldrich and Roche. TRI from Roche was better...

-test tube-

QUOTE (test tube @ Mar 30 2007, 07:08 PM)

QUOTE (Garden @ Mar 17 2007, 11:09 AM)

Thanks for your instuction! I extracted once more yesterday.Could you think the total RNA is degraded? The second picture is 6% PAGE.

You made PAGE for RNA analysis?

I dont know if this could help but we had in our lab TRIreagent from 2 companies Sigma-Aldrich and Roche. TRI from Roche was better...

I want to know if TRI from Roche is suitable for miRNA isolation. In other word, dose this reagent can extract small RNA from tissue? Thanks for your help, 'test tube' .

-Garden-

Can you briefly mention the protocol you follow?

-polsum-

QUOTE (polsum @ Apr 4 2007, 04:34 AM)

Can you briefly mention the protocol you follow?

I can extract correct total RNA from Drosophila using the same protocol, but can't extract good RNA from my species. I don't know why. So I doubt Trizol(Invitrogen) is not suitable for my species. I don't know whether the result will be good if I use another Tri reagent.

-Garden-