[Histonet] RNA isolation form stained slides

[Sue Hunter]

Have you tried to extract the RNA from the tissue without the stain- just to test your method?  I am not familiar with the mechanics of the stain but you might be denaturing the RNA.  Also are you sure there is enough message in the area you are trying to extract to get enough RNA?  Maybe you need more material.

Sue Hunter, Supervisor
Advanced Diagnostics
Beaumont Health System
Royal Oak MI
248-898-5146
shunter <@t> beaumont.edu


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Vanessa Orsini
Sent: Friday, November 02, 2012 5:43 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RNA isolation form stained slides







Hello,

I need to extract RNA for a RT-PCR after Laser MicroDissection on xGal stained slides.

I tried using sections from unfixed frozen organs. I fixed the sections in EtOH70% for 10min and then I stained them with xGal for 3h at 37°C.
After air drying I cut out with the LCM and extract RNA with the PicoPure kit from Applied Biosystem. So far I didn't manage to get enough RNA. 

I tried to add RNase inhibitors to all the solutions but it didn't help.

 

Any idea/suggestion?

Do someone think it would be better to do a LacZ antibody staining on FFPE sections and extract RNA with an appropriate kit? The RNase would they be less active?

 

Thank in advance for any help you can give me J Vanessa

 		 	   		  _______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet