Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay |
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Authors: | C Mittmann Ursula Münstermann Joachim Weil Michael Böhm Stefan Herzig Christoph Nienaber Thomas Eschenhagen |
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Institution: | Pharmakologisches Institut, Universit?ts-Krankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany, DE Innere Medizin, Klinik III, Universit?t K?ln, Cologne, Germany, DE Pharmakologisches Institut, Universit?t K?ln, Cologne, Germany, DE Medizinische Klinik, Abteilung Kardiologie, Universit?ts-Krankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany, DE
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Abstract: | End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These
changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology,
clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the
quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the
RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium
comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts
with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the β1-adrenoceptor, the stimulatory G protein α-subunit (Gsα), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), β-myosin heavy chain (β-MHC), and the atrial natriuretic
peptide (ANP). We extracted 10.7±2.1 μg total RNA from three myocardial biopsies taken in vitro. All of the six genes were
measurable in duplicate in a total of 7 μg RNA. mRNAs of β1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively,
whereas β-MHC and Gsα mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific
and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsα levels obtained by Northern blot analysis with 7.5 μg total RNA showed a good correlation between the two methods. The RNase
protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies.
Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial
ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.
Received: 12 May 1997 / Accepted: 8 September 1997 |
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Keywords: | Heart failure Human RNase protection assay Myocardial biopsies Gene expression |
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