Viral RNA extraction - Formalin fixed & Paraffin embended tissue (Sep/19/2005 )

Dear friend,

I facing a problem to extract viral RNA out from a formalin fixed, paraffin embended viral infected tissue. However after extraction, the ectracted RNA is either not pure (ratio 1.27-1.35) or RNA concentration too little (6 ug/ml).
I run with a RT-PCR togather with positive control (viral culture), positive control got band, my sample from paraffin got no band.

Any idea? Any suggestion will be greatly appreciated.

Thank you.

Yongyk

Dear freind,

This is how I treat my sample.
First 15 section of viral infected tissue was put into a 1.5ml tube.

1200ul of xylene was added into the tube to deparaffin the sample.
The sample was then incubate in water bath at 55oC for 10 min.

Spin at 14000 rpm for 10 min. Discard the supernatant by pipetting.

This xylene washing procedure was carried out for twice.

The 1000ul absolute ethanol was added to was away xylene recidue, spin at 14000 rpm for 10 min and discard the supernatant by pipetting (x2).

Then proceed with standard Trizol protocol.



Friends, please kindly offer me a good protocol or help me to improve my protocol.
Any help will be greatly appreciated.
Thank you very much


Best regards
Yongyk

Hello
I don’t have idea for ur problem. But how do u prepare Formalin fixed & Paraffin embended tissue. Kindly give me protocol.

Thank you,
Suguna

Dear Suguna,

I do not prepare those formalin fixed, paraffin emebnded tissue. Those tissue are provided by pathologist. Basically, they had infected some mice with Enterovirus 71, and they found out that those mice mascles tissue were highly infected. So they past those mascles tissue to me hopping that I can tell them the viral load.

However, when I done my RNA extraction, I only have around 6ug/ml of RNA out of 25mg of tissue sample and the purity ratio is 1.2 or 1.3. When I proceed for my RT-PCR, got no band come out.

In the first place, 25mg of tissue should not get so little of RNA.
So is there any way to improve my paraffin fix tissue's RNA extraction?

Thank you.

yongyk

Dear yongyk,

Thank you for your reply. I don't have any experience of isolation of RNA from paraffin embedded tissues. I am attaching some links below, that may be useful for you.


http://www.mlpa.com/RNADNA%20extraction.htm
http://www.cmj.org/information/full.asp?id=621

http://www.pathtech.com.au/files/011X0I7I9...urification.pdf


RNA extraction from formalin-fixed and paraffin-embedded tissues.
Stanta G, Bonin S, Perin R.
Methods Mol Biol. 1998;86:23-6.



good luck
suguna

Dear Suguna,

Thank you very much.
When I look at those web site, it greatly improve my understanding about RNA extraction from paraffin enbeded tissue.

I shall up date you guy when I got it.

Thank you very much



Best regards
Yongyk

Dear yongyk,

Regarding to the RNA extraction for FFPE tissue, Mark Dorris had done some detail study on
REVERSING THE EFFECTS OF FORMALIN FIXATION (PhD thesis, 1999)

Take a look on this webside:
http://www.nematodes.org/protocols/formalin/formalin.html

Hope this is useful to you.

Dear yongyk,
I have some news for you.
Try out this product Optimum FFPE kit from Ambion.

Have you got any thing yet so far?
Since you descript you work in detail, so I am repeating yours work with my own mice and virus.
The mice tissues were fix in formalin for two weeks, paraffin embeded.
I used 5um x 50 sections, so i will give me an average of 20 to 25mg tissue.
I used the above kit to extract RNA and I manage to get 46.5ug/ml and A260:A280 = 2.4.

Good luck

Hi there,

I personally would recommend RNAqueous -4PCR kit for isolation of RNA (AMBION KIT). For me it usually yeild RNA of around 1.8-2.1 (260/280) and it does function well for applications such as RT-PCR.

NB: You have to note that RNA is very unstable, so to improve your results, I would recommend reverse trancribing RNA to cDNA immediately after extraction. Altenatively store your RNA @-80oC