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      Reliability of T7-Based mRNA Linear Amplification Validated by Gene Expression Analysis of Human Kidney Cells Using cDNA Microarrays

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          Abstract

          Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp<sup>®</sup> amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and two rounds of linear RNA amplification, respectively, the amplified RNAs were co-hybridized to cDNA microarrays. Linearity and reproducibility of the individual experiments were then assessed by calculating the Pearson correlation. The intra-amplification consistency showed a correlation of 0.968 for the first round, 0.907 for the second round and 0.912 for two successive rounds of amplification. If the first round was compared to unamplified material, r was 0.925. The second round amplification yielded a correlation of 0.897 if compared to unamplified mRNA. Two rounds of amplification starting from 200 pg of mRNA compared to unamplified material resulted in a correlation of 0.868. These results indicate that linear amplification using RiboAmp<sup>®</sup> kit yields amplified RNA with a high degree of linearity and reproducibility.

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          High-fidelity mRNA amplification for gene profiling.

          The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.
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            CELL SAMPLING: Laser Capture Microdissection: Molecular Analysis of Tissue

             R Bonner (1997)
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              Author and article information

              Journal
              NEE
              Nephron Exp Nephrol
              10.1159/issn.1660-2129
              Cardiorenal Medicine
              S. Karger AG
              1660-2129
              2004
              July 2004
              17 November 2004
              : 97
              : 3
              : e86-e95
              Affiliations
              aDepartment of Nephrology, University Hospital of Innsbruck, Innsbruck, bEmergentec Business Analytics, Vienna, and cTyrolean Cancer Research Institute, Innsbruck, Austria; dDepartment of Nephrology, Stanford University Medical School, Stanford, Calif., USA
              Article
              78642 Nephron Exp Nephrol 2004;97:e86–e95
              10.1159/000078642
              15292679
              © 2004 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: 2, Tables: 4, References: 13, Pages: 1
              Product
              Self URI (application/pdf): https://www.karger.com/Article/Pdf/78642
              Categories
              Original Paper

              Cardiovascular Medicine, Nephrology

              RNA, Gene expression, Microarrays, Kidney, Amplification

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