Re: [Histonet] RE: Out of my comfort zone...
We use a heat block during digestion. There is less chance of contamination with a heat block than with a water bath. ... and no we don't use antigen retrieval solution for this! We use a Qiagen kit too. Mark On Wed, Apr 3, 2013 at 9:11 AM, Helen Fedor wrote: > We have been using store bought gallons of distilled water in our water > baths. This water has been boiled so enzyme activity should be absent. > > > Helen > > 410.614.1660 > > http://tmalab.jhmi.edu/ > http://prostatebiorepository.org/ > > > > Helen > > -Original Message- > From: histonet-boun...@lists.utsouthwestern.edu [mailto: > histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sue Hunter > Sent: Wednesday, April 03, 2013 10:55 AM > To: Sarah Dysart; histonet@lists.utsouthwestern.edu > Subject: [Histonet] RE: Out of my comfort zone... > > Hello > Just wanted to add one more thing - we actually use a dedicated pyrex dish > (maybe 6x10 inches) for our water bath for RNA sections. We use warm tap > water, but you can put it in the microwave for a short bit if it needs to > be warmer. You can spray the dish with RNAse away and wipe before filling > with water. > Sue > > -Original Message- > From: histonet-boun...@lists.utsouthwestern.edu [mailto: > histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sarah Dysart > Sent: Tuesday, April 02, 2013 5:36 PM > To: histonet@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@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Out of my comfort zone...
We have been using store bought gallons of distilled water in our water baths. This water has been boiled so enzyme activity should be absent. Helen 410.614.1660 http://tmalab.jhmi.edu/ http://prostatebiorepository.org/ Helen -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sue Hunter Sent: Wednesday, April 03, 2013 10:55 AM To: Sarah Dysart; histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Out of my comfort zone... Hello Just wanted to add one more thing - we actually use a dedicated pyrex dish (maybe 6x10 inches) for our water bath for RNA sections. We use warm tap water, but you can put it in the microwave for a short bit if it needs to be warmer. You can spray the dish with RNAse away and wipe before filling with water. Sue -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sarah Dysart Sent: Tuesday, April 02, 2013 5:36 PM To: histonet@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@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Out of my comfort zone...
Hello Just wanted to add one more thing - we actually use a dedicated pyrex dish (maybe 6x10 inches) for our water bath for RNA sections. We use warm tap water, but you can put it in the microwave for a short bit if it needs to be warmer. You can spray the dish with RNAse away and wipe before filling with water. Sue -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sarah Dysart Sent: Tuesday, April 02, 2013 5:36 PM To: histonet@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@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Out of my comfort zone...
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 shun...@beaumont.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sarah Dysart Sent: Tuesday, April 02, 2013 5:36 PM To: histonet@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@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
