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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Editorial Board Member: Gillery, Philippe / Kazmierczak, Steven / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Whitfield, John B.

12 Issues per year

IMPACT FACTOR 2013: 2.955
Rank 5 out of 29 in category Medical Laboratory Technology in the 2013 Thomson Reuters Journal Citation Report/Science Edition

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Evaluation of RNA Isolation Methods and Reference Genes for RT-PCR Analyses of Rare Target RNA

Christine Mannhalter / Daniela Koizar / Gerlinde Mitterbauer

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 38, Issue 2, Pages 171–177, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2000.026, June 2005

Publication History

Published Online:
2005-06-01

Abstract

Reverse transcription polymerase chain reaction (RTPCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol™, RNAzol™, FastTube™ reagent). RNA yield was slightly higher with RNAzol™ than with TRIzol™ as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube™ reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol™ and TRIzol™. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol™, and 1 BCR-ABL-positive (specific for translocation t [9; 22]) cell among 2×104 normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol™, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

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