In order to complicate things, Does anyone have experience in FISH plus Flow in the area of microorganisms, bacteria or yeast, or know someone who has? Thanks, Joerg -------------------------------------- Dr. Joerg Ueckert Unilever R&D Vlaardingen Food Processing Group Postbus 114 3130 AC Vlaardingen Netherlands Tel. +31-10-4605196 Fax +31-10-4605188 Joerg.ueckert@unilever.com -------------------------------------- -----Original Message----- From: dkaplan@flow-amp.com [SMTP:dkaplan@flow-amp.com] Sent: Tuesday, January 15, 2002 10:27 PM To: Cytometry Mailing List Subject: FISH and flow cytometry << File: flowamp-42-eber.ppt >> << File: flowamp43-eber.ppt >> << File: Attachment >> Cheryl, Concerning fluorescent in situ hybridization for the detection of mRNA species by flow cytometry, sensitivity is a major problem. In situ RT-PCR has not worked well. We have used our enzymatic amplification staining (EAS) technology for this problem and have achieved excellent results (attached 2 powerpoint figures). Flow-Amp's EAS kit for intracellular antigens with FITC conjugated primary antibodies was used with substitution of a FITC conjugated cDNA probe for Epstein-Barr Virus EBER1 RNA instead of an antibody. The difficulty in the procedure was getting the hybridization conditions right but the results were impressive. David David Kaplan, MD, PhD Professor of Pathology Case Western Reserve University Co-Founder, Flow-Amp Systems Hello All, I've had a query from one of our facility users regarding the ability of flow cytometry to detect single FISH probes. The most recent posting from the group that I can find is from 1996 and I'm wondering if everyone has simply decided that flow is not sensitive enough for this. It was mentioned that incorporation of digoxigenin into the PCR reaction apparently increased single to noise ratio, but is it really enough to enable detection? Any input would be greatly appreciated. Thanks, Cheryl
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