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Impact of formalin-fixation and paraffin-embedding on the ration between mRNA copy numbers of differently expressed genes
- Art
- Zeitschriftenartikel
- Autor/en
- Smolinski D v; Leverköhne I; Samson-Himmelstjerna G v; Gruber A D
- Quelle
- Histochemistry Cell Biology, 124, 2005, 177-188
- Sachgebiet
- Pathology
- Tierart/en
- General
- Schlagwörter
- quantitative gene expression, real-time reverse transcriptase polymerase chain reaction, formalin fixation, paraffin embedding
- Kontakt
- Institut für Tierpathologie
Robert-von-Ostertag-Str. 15
14163 Berlin
Tel.+49 30 838 62450 Fax.+49 30 838 62522
email:pathologie(at)vetmed.fu-berlin.de - Link
- Impact of formalin-fixation and paraffin-embedding on the ration between mRNA copy numbers of differently expressed genes
- 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 ration 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 up to 20 years hat no additional effects.
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