[Histonet] RE: Out of my comfort zone...

[Sue Hunter]

Sarah
The proteinase K does a lot more than break the formalin linkages.  To isolate the RNA or DNA you have to break up the cell membranes, nuclear membranes and all the other proteins in the cellular matrix to isolate the nucleic acids.  I don't think an antigen retrieval solution will do any of that.  We use a kit from Qiagen that is very easy to use.   Also, if you haven't done much RNA work, remember that there are RNAses everwhere.  Wear gloves, wipe down your microtome with RNAse away or some other such product. Use a clean blade. Discard the first couple of cuts from your block.  Too many fingers have touched them.  We usually cut 10um sections for our extractions.  Use clean water in your waterbath - fresh just for your RNA tissues.  We keep slide boxes separate for RNA work so bare fingers don't touch them. As for how many sections - it depends on how much message you will be looking for.  You will have to try your method to find out.    If you cut curls for extractions, we use 10um curls.   Use disposable plastic tubes as these are mostly RNAse free.  We routinely isolate sufficient quantities of good RNA from FFPE tissues, but you still need to use good RNA technique.
If you are making up your own master mixes and primer mixes, be sure to use RNAse free water.
Good luck
Sue

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 Sarah Dysart
Sent: Tuesday, April 02, 2013 5:36 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Out of my comfort zone...

So...I have been asked to do some micro-dissection on some slides and then do downstream RT/PCR on them.  My molecular knowledge doesn't go much out of the world of IHC so...here is my question...

Has anyone ever substituted Citrate Buffer pH6 (or whatever HIER solution you are using) for proteinase K for use in RNA isolation and then later PCR?  Does this work?  The main question is will the HIER step take off the formalin linkage from the nucleic acids, or just the protein?

One last thing is what else goes into these solutions other than Citrate Buffer and Tween?  I haven't made it up in forever, I have just been ordering it from companies...I know...lazy...

Thanks

Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP) Histotechnologist Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912

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