5 replies to this topic

[ColoSarah]

Hi all! I was hoping to get some feedback on issues I am having with isolating high quality RNA from guinea pig lung. I am aware that the lung is #3 in highest RNase activity, so from the get go I am going to have a tough time. Here are the following protocols I have tried with no success:

Excise tissue
Snap freeze
Homogenize in TRIzol
Snap freeze
Follow TRIzol isolation protocol

Excise tissue
Cut with razor blade
Put into RNA Later
Freeze at -20 for 1 day
Homogenize in TRIzol

Excise tissue
Put into iRPMI
Perform enzymatic digestion with collegenase and DNase
Trypan blue to access viability (40-65%)
TRIzol cells

All of the above protocols have not worked. I am hoping to use this lung RNA for qPCR, but all of my RIN values from the agilent bioanalyzer are 2.3-2.5 out of 10. Very poor. It appears I just have fragments of RNA. I know this collection process needs to be down very rapidly and that RNase activity is very quick. I am pretty certain the degradation is occurring from the time of collection to my isolation. My isolation procedures are sound. I have been able to get great RNA out of Thp-1 cells that are processed at the same time as my lung tissue.

Any feedback on this issue would be super helpful.

Thanks!

[bob1]

Your first protocol should be the best.  If it is frozen in LN2 or on dry ice, you can probably grind the sample into powder before adding to Trizol.

Have you looked at the RNA on a gel (rather than just the bioanalyser) and/or determined the concentration by spectrophotometry?

[ColoSarah]

It is snap frozen in liquid nitrogen. I use a tissue tearor to homogenize the sample while in TRIzol. I haven't tried grinding the sample into a fine powder.

I have run all of my samples on RNA dentauring gels. I always get a low molecular weight smear of fragmented RNA. Just for kicks they always get run on the bioanalyzer and all come back as crappy as the RNA gel determined. The A260/280 ratios are fantastic. 2.0-2.2 That ratio just tells me that I have nucleic acids (not proteins, etc.) so it isn't of great help.

Edited by ColoSarah, 28 July 2011 - 12:31 PM.

[bob1]

OK, cool; it sounds like you know what you are doing.  I know some people working on the pancreas, who found that they had to use the RNA within a week of extraction, but it sounds like your problem is earlier than that.

How low a MW is the smear you mentioned?  It may be that you have the mRNA that you need somewhere in there.

Have you tried column extractions, which are typically very fast?

Have you tried raising the amount of trizol relative to tissue?

[JoanaSantos]

Hello everyone!
I'm having some similar issues that you described with isolation high quality RNA.
My samples are from hip arthroplasty and although the A260/280 ratios are really great (1,92; 2,01 and 2,03), when i run them into a gel (agarose) there are no bands.


I have tried the TRizol isolation protocol and the RNA kit from invitrogen with no sucess.




Any feedback in this issue would be so helpful.

Thank you!

[Trof]

I myself am just fighting my way through RNA isolation too, but few teoretical notes:

ColoSarah, on 27 July 2011 - 01:38 PM, said:

Excise tissue
Snap freeze
Homogenize in TRIzol
Snap freeze
Follow TRIzol isolation protocol

As bob1 wrote, grinding to smaller pieces while still frozen before adding Trizol should help quicker homogenisation. AFAIK the more time it takes the Trizol to lyse the tissue completely the more time has the RNAase to screw your sample. Also after adding Trizol and homogenisation with pipetting the sample in Trizol should be left at RT for 5 minutes to complete the lysis.

ColoSarah, on 27 July 2011 - 01:38 PM, said:

Excise tissue
Cut with razor blade
Put into RNA Later
Freeze at -20 for 1 day
Homogenize in TRIzol

Samples in RNAlater should be left for one day at 4 deg prior to freezing to -20, the manual says, to completely soak the tissue. If you use it the next day I see no real reason for freezing it as it's OK at 4 deg, but probably you test it for longer storage.
Even when you use RNAlater the problem stays the same as in the first protocol, somehow disrupt the tissue to smaller pieces that will lyse in Trizol immediately. Maybe you can snap freeze them and grind or just cut the RNAlater-stored sample into small pieces with a scalpel while keeping it soaked.