From owner-recom@hgmp.mrc.ac.uk Mon Apr 7 04:24:04 2003 Return-Path: X-Original-To: recom-outgoing Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with SMTP id DDC307D0DF for ; Mon, 7 Apr 2003 04:24:03 +0100 (BST) X-Original-To: recom-list@hgmp.mrc.ac.uk Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 7FADC7D14D for ; Mon, 7 Apr 2003 04:24:03 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 60001) id 82CAA7D0DF; Mon, 7 Apr 2003 04:24:02 +0100 (BST) To: recom@net.bio.net Newsgroups: bionet.molbio.recombination From: jkhattra@bcgsc.bc.ca ("Jaswinder Khattra") Subject: Mouse brain total RNA gel and Agilent Date: 17 Mar 2003 22:57:57 -0000 Message-Id: <20030407032402.82CAA7D0DF@mercury.hgmp.mrc.ac.uk> Sender: owner-recom@hgmp.mrc.ac.uk Precedence: bulk Hello, We have been looking at the integrity of total RNA isolated from mouse embryo brain regions. These have been harvested by established methods, such as using RNALater or liquid nitrogen, followed by extraction with Trizol. Total RNA is routinely analyzed on an Agilent Bioanalyzer and using denaturing agarose gels (EtBr staining). We know what intact total RNA should look like by both methods. What is peculiar about many of these samples is that the rRNA intensity ratios are about equal (not 2:1, LSU:SSU) , but there is little indication of degradation i.e. no smearing in the gel; no hump in the Agilent Bioanalyzer profile left of the rRNA bands. Is it possible that the LSU rRNA is intact, but it's secondary structure is such that the intercalation of EtBr or binding of fluorescent dye is not representative of the actual amount relative to SSU rRNA? What also does not make sense is that if the LSU band is degraded due to ribonuclease activity, other RNA species ought to be degraded as well (unless nucleases are LSU sequence- or conformation-specific). Any suggestions to resolve this - other than amplifying out increasingly large fragments by RT-PCR. Thank you. Jas Mr. Jaswinder Khattra Production Coordinator Gene Expression Lab Genome Sciences Centre BC Cancer Agency 600 West 10th Avenue Vancouver BC Canada V5Z4E6 > (604) 877-6000 ext.3267, lab 3266 > (604) 877-6085 fax > jkhattra@bcgsc.ca > www.bcgsc.ca ===== Moderated bionet.genome.gene-structure __________________________________________________ Yahoo! Plus For a better Internet experience http://www.yahoo.co.uk/btoffer