RNA handling for RT-PCR? - (Jul/23/2007 )

Hi

I hav a question: if I am to do the real time PCR form animal tisse samples (human blood in my case)..I immediately process the blood to get my RNA ( i have been using RNeasy micro kit) and then i tend to quickly store my RNA at -20 degrees.

Is it the right way of doing it? or am i supposed to quickly conver this RNA to cDNA and then better store it????

I also need to know how necessary is it to go for RNA quality check in such case...do i have to do it every time, we do not have any bioanalyzer in our lab and i have limited RNA available so hate wasting on these checks!!!! The worst part is I have very limited amounts of RNA after extraction from the samples I get and i don't think i could afford to waste any!!

please suggest.

Rits

-rits-

well RNA should be stored at -80.
For checking quality, a brief spectrum ranging from OD230 to OD 290 should tell you some informations. Should be done every time.
For your extraction procedure, i would advise you to do 3-4 extractions, all at diferent times, and check on bioanalyser in order to tell your extraction procedure is ok.
Otherwise, you may run 10µg on 10% denaturing gel and see the profine.
i attached 3 pictures ranging from bad to high quality(extracts from 293cells). i prefer tis method to check a the agarose one as it's more sensitive due to the fact ou see all small rnas and degradation forms small RNAs)
finally, extract and quantify your RNAs, and then aliquot them in order to avoid 2 thaws for RTPCR.

-fred_33-

QUOTE (fred_33 @ Jul 23 2007, 04:52 PM)

Otherwise, you may run 10µg on 10% denaturing gel and see the profine.
i attached 3 pictures ranging from bad to high quality(extracts from 293cells). i prefer tis method to check a the agarose one as it's more sensitive due to the fact ou see all small rnas and degradation forms small RNAs)
finally, extract and quantify your RNAs, and then aliquot them in order to avoid 2 thaws for RTPCR.

Hi,
I resumed this old thread as I need some advices. I'm going to work with RNA for the very first time.

Let's see if I got the idea...
The idea beneath the check on agarose gel is that total RNA is mainly composed by ribosomal RNA. So you are expecting to find a defined profile due to the presence of the RNA from ribosomal subunits. If you have smears, it means that there is a lot of degradated RNA that has run between the ribosomal bands.
Am I right?

Thanks for helping!
ILA

-ila-

there are some people suggest to me that, to run RNA on agarose, use 1% gel, use 120V, run only 20 minutes.

also, RNA MUST store at -80C.

-sanjiun81-

QUOTE (sanjiun81 @ Jan 29 2008, 09:42 AM)

there are some people suggest to me that, to run RNA on agarose, use 1% gel, use 120V, run only 20 minutes.

also, RNA MUST store at -80C.

Thanks sanjiun!
I'm going to perform my first extraction this afternoon.
Wish me luck...

-ila-