Publikationsdatenbank

Impact of Formalin-fixation and Paraffin-embedding on the Ratio between mRNA Copy Numbers of Differently Expressed Genes (2005)

Art

Zeitschriftenartikel / wissenschaftlicher Beitrag

Autoren

von Smolinski, Dorthe

Leverkoehne, Ina

von Samson-Himmelstjerna, Georg

Gruber, Achim D

Quelle

Histochemistry and cell biology

Bandzählung: 124

Heftzählung: 2

Seiten: 177 – 188

ISSN: 0948-6143

Sprache

Englisch

Verweise

Pubmed: 16049695

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
14163 Berlin
+49 30 838 62450
pathologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Several studies have shown that specific mRNA sequences can be successfully detected in formalin-fixed, paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction (RT-PCR). Here, we test the hypothesis that gene expression levels can be accurately quantified in formalin-fixed, paraffin-embedded tissues by determining the ratio between the copy number of the mRNA molecule of interest and the mRNA copy number of a so-called housekeeping gene. The mRNA copy numbers of the variably expressed multiple drug resistance gene (MDR)-1 and four housekeeping genes (hypoxanthine phosphoribosyl-transferase-1, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and elongation factor-1a) were quantified by real-time-quantitative RT-PCR before and after formalin-fixation and paraffin-embedding of 576 tissue samples (heart, kidney, spleen, liver) from three beagle dogs. The results indicate that fixation and embedding drastically altered the ratios between the different mRNA copy numbers and that the relative expression levels of MDR-1 per any of the housekeeping genes were artificially increased or decreased up to more than tenfold. It would thus appear questionable to normalize quantitative expression data from fixed and embedded tissues by using housekeeping genes as reference. In contrast, tissue autolysis of up to 24 h and long-term storage of embedded tissues of up to 20 years had no additional effects.