Collin, I would suggest sorting directly into RLT buffer (Qiagen-RNeasy Mini kit) if RNAlater is not working for you. In my experience....For general purpose RNA isolation, cell sorting followed by centrifugation and removal of supernatant prior to lysis with the appropriate volume of RLT buffer is the best method for preserving RNA quality. Years ago, we sorted erythrocyte precursors directly into RLT buffer and found that this was the only way to obtain enough decent quality RNA for expression profiling. How is your "to sort" population viability at the beginning and end of the sort? This may be an indication of the quality of RNA you are going to get in the end. Best of luck. Chris Chris Groves Scientist I Autoimmunity and Respiratory Diseases MedImmune One MedImmune Way Gaithersburg, MD 20878 301-398-5586 grovesc@medimmune.com -----Original Message----- From: Collin White [mailto:ccwhite@u.washington.edu] Sent: Monday, May 08, 2006 4:17 PM To: cyto-inbox Subject: [Fwd: Sorting macro's from whole liver populations and RNA quality] addition The cell population was also counter stained with PI to separate out necrotic cells. -------- Original Message -------- Subject: Sorting macro's from whole liver populations and RNA quality Date: Mon, 08 May 2006 12:49:59 -0700 From: Collin White <ccwhite@u.washington.edu> To: cyto-inbox Hi all, I am writing for any help or insight on preserving RNA quality in macrophages sorted for micro array from whole perfused mouse liver populations. Have already sorted on a variety of markers in the Hepatocytes with no real problems but am having trouble with the Kupffer's/Marcro's. I am using PE F4/80 as my marker. I have tried sorting in RNA later (Qiagen), 50/50 RNA later/media and just media. After sorting120,000 cells I spin down the sample resuspend in RNA lysis buffer and freeze in the -80 for RNA purification and quality (bioanalysis) the next day. The best quality I can get is from just sorting in media but there is still appreciable degradation. Any help would be appreciated. Collin Collin White Manager, CEEH Analytical Cytology Lab Department of Environmental and Occupational health Sciences University of Washington -- Collin White Research Scientist University of Washington 4225 Roosevelt Way NE #100 DEOHS Seattle, WA 98105 Ph 206-616-4982Received on Wed May 10 13:38:00 2006
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