tag:blogger.com,1999:blog-6011966735431330184.post5028976030762981049..comments2023-10-17T22:37:35.235+07:00Comments on Clinical genetics, Medical genetics and molecular genetics: RNA isolation protocol (1)Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.comBlogger5125tag:blogger.com,1999:blog-6011966735431330184.post-23441647990856493332007-11-18T18:07:00.003+07:002007-11-18T18:07:00.003+07:005. Cover open tubes with Paraffin and pierce sever...5. Cover open tubes with Paraffin and pierce several times with a fine needle. Dry pellet briefly under vacuum or dry on bench. Store dry RNA < or equal 2 months at – 70 degree celcius or below, or dissolve in 20 microlitre DEPC-treated water if the assay will carried out immediately.Dr Prasit Phowthongkumhttps://www.blogger.com/profile/12398709883713733111noreply@blogger.comtag:blogger.com,1999:blog-6011966735431330184.post-72883454105141779372007-11-18T18:07:00.002+07:002007-11-18T18:07:00.002+07:004. Add 300 microlitre RNA isolation solution II to...4. Add 300 microlitre RNA isolation solution II to pellet and vortex to suspend. Microcentrifuge briefly at maximum speed. Add 600 microlitre of 100% ethanol and mix. Incubate > or equal 1 hr at -20 degree celcius. Microcentrifuge 15 min at high speed, 4 degree celcius. Discard supernatant. Wash pellet with 0.5 ml of 70% ethanol. Discard ethanol. If pellet dislodges from bottom of tube, microcentrifuge 5 min at high speed, 4 degree celcius before removing ethanol.Dr Prasit Phowthongkumhttps://www.blogger.com/profile/12398709883713733111noreply@blogger.comtag:blogger.com,1999:blog-6011966735431330184.post-73088983465952429122007-11-18T18:07:00.001+07:002007-11-18T18:07:00.001+07:003. remove 0.4 ml of aqueous (upper) phase to a cl...3. remove 0.4 ml of aqueous (upper) phase to a clean 1.5 ml tube that contains 0.8 ml of 100% ethanol, taking care not to disturb interface. Incubate > 1 hr at -20 degree celcius. Microcentrifuge 15 min at maximum speed, 4 degree celcius. Discard supernatant.Dr Prasit Phowthongkumhttps://www.blogger.com/profile/12398709883713733111noreply@blogger.comtag:blogger.com,1999:blog-6011966735431330184.post-62328418672182305622007-11-18T18:07:00.000+07:002007-11-18T18:07:00.000+07:002. Add 0.5 ml RNA isolation solution I to cell pel...2. Add 0.5 ml RNA isolation solution I to cell pellet. Disrupt cells by repeated aspiration. Add 0.05 ml of 2 M sodium acetate. Vortex and microcentrifuge briefly at maximum speed. Add 0.5 ml buffered phenol. Vortex and microcentrifuge. Add 0.1 ml of 24:1 chloroform/isoamyl alcohol. Vortex and microcentrifuge 15 min at maximum speed 4 degree celcius.Dr Prasit Phowthongkumhttps://www.blogger.com/profile/12398709883713733111noreply@blogger.comtag:blogger.com,1999:blog-6011966735431330184.post-52690400416108184892007-11-18T18:06:00.000+07:002007-11-18T18:06:00.000+07:001. Wash mononuclear cell pellete once with HBSS wi...1. Wash mononuclear cell pellete once with HBSS without Ca or Mg. Resuspened cells in HBSS and divide among three 1.54 ml microcentrifuge tubes. Microcentrifuge 2 min at 100 X g to pellet cells. Discard supernatant. Use immediately or store < or equal 4 months at -70 degree celcius. Use one tube to prepare RNA and the other two repeat assay.Dr Prasit Phowthongkumhttps://www.blogger.com/profile/12398709883713733111noreply@blogger.com