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      A Comparative Kinetic RT/-PCR Strategy for the Quantitation of mRNAs in Microdissected Human Renal Biopsy Specimens

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          Abstract

          Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

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          Author and article information

          Journal
          EXN
          Nephron Exp Nephrol
          10.1159/issn.1660-2129
          Cardiorenal Medicine
          S. Karger AG
          1660-2129
          1998
          December 1998
          06 November 1998
          : 6
          : 6
          : 563-567
          Affiliations
          Division of Nephrology, Institute of Internal Medicine, University of Padova, Italy
          Article
          20572 Exp Nephrol 1998;6:563–567
          10.1159/000020572
          © 1998 S. Karger AG, Basel

          Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

          Page count
          Figures: 3, References: 10, Pages: 5
          Product
          Self URI (application/pdf): https://www.karger.com/Article/Pdf/20572
          Categories
          Original Paper

          Cardiovascular Medicine, Nephrology

          β-Actin, RT/-PCR, Glomeruli, G3PDH, Kidney biopsy

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