From: Mark A. KuKuruga (kukuru@umich.edu)
Date: Tue Oct 27 1998 - 12:23:20 EST
Andreas Simm wrote: > Hello Harry > we also tried to get RNA from sorted cells. We did not get RNA from > paraformaldehyd fixed cells. Andreas,Can't place hands on specific reference, but I do know several reports claim successful mRNA extraction from PFA fixed cells. Probably depends on fixing technique and target message. This may be an example: Longley J. Ding TG. Cuono C. Durden F. Crooks C. Hufeisen S. Eckert R. Wood GS. Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells. Journal of Investigative Dermatology. 97(6):974-9, 1991 Dec. > On the other hand, we had good results using a 70% ethanol fixation. We > stained the cells after fixation for example > with a FITC-conjugated anti PCNA antibody and sorted the positive versus the > negative cells. Thereafter, we isolated > the RNA or used the sorted the cells directly into PCR tube for a following > PCR. This will be published in Cardiovascular > Research, I can send you a preprint, if you want. I also have several users interested in this technique . . . I would appreciate a reprint as well. > An additional paper exists from the group of Dr. Radbruch, using a > ethanol/acedic acid combination for the fixation (in Cytometry, I don`t know > the exact citation). FYI . . . Esser C. Gottlinger C. Kremer J. Hundeiker C. Radbruch A. Isolation of full-size mRNA from ethanol-fixed cells aftercellular immunofluorescence staining and fluorescence-activated cell sorting (FACS). Cytometry. 21(4):382-6, 1995 Dec 1. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry 7416 CCGC 1500 East Medical Center Drive Ann Arbor, MI 48109-0946 U.S.A.
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