Abstract
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.
Similar content being viewed by others
References
Abrahamsen HN, Steiniche T, Nexo E, Hamilton-Dutoit SJ, Sorensen BS (2003) Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. J Mol Diagn 5:34–41
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell. Garland Science, New York
Ben-Ezra J, Johnson DA, Rossi J, Cook N, Wu A (1991) Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J Histochem Cytochem 39:351–354
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Czworkowski J, Moore PB (1996) The elongation phase of protein synthesis. Prog Nucleic Acid Res Mol Biol 54:293–332
Endicott JA, Ling V (1989) The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58:137–171
Fitzpatrick R, Casey OM, Morris D, Smith T, Powell R, Sreenan JM (2002) Postmortem stability of RNA isolated from bovine reproductive tissues. Biochim Biophys Acta 1574:10–14
Foss RD, Guha-Thakurta N, Conran RM, Gutman P (1994) Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls. Diagn Mol Pathol 3:148–155
Foss DL, Baarsch MJ, Murtaugh MP (1998) Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues. Anim Biotechnol 9:67–78
Ginn PE (1996) Immunohistochemical detection of P-glycoprotein in formalin-fixed and paraffin-embedded normal and neoplastic canine tissues. Vet Pathol 33:533–541
Goldsworthy SM, Stockton PS, Trempus CS, Foley JF, Maronpot RR (1999) Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcin 25:86–91
Gruber AD, Levine RA (1997) In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha). Histochem Cell Biol 107:411–416
Gruber AD, Moennig V, Hewicker-Trautwein M, Trautwein G (1994) Effect of formalin fixation and long-term storage on the detectability of bovine viral-diarrhoea-virus (BVDV) RNA in archival brain tissue using polymerase chain reaction. Zentralbl Veterinarmed B 41:654–661
Impraim CC, Saiki RK, Erlich HA, Teplitz RL (1987) Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun 142:710–716
Kingsbury AE, Foster OJ, Nisbet AP, Cairns N, Bray L, Eve DJ, Lees AJ, Marsden CD (1995) Tissue pH as an indicator of mRNApreservation in human post-mortem brain. Brain Res Mol Brain Res 28:311–318
Koopmans M, Monroe SS, Coffield LM, Zaki SR (1993) Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. J Virol Methods 43:189–204
Lehmann U, Kreipe H (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25:409–418
Leverkoehne I, Horstmeier BA, von Samson-Himmelstjerna G, Scholte BJ, Gruber AD (2002): Real-time RT-PCR quantitation of mCLCA1 and mCLCA2 reveals differentially regulated expression in pre- and postnatal murine tissues. Histochem Cell Biol 118:11–17
Lewis F, Maughan NJ, Smith V, Hillan K, Quirke P (2001) Unlocking the archive-gene expression in paraffin-embedded tissue. J Pathol 195:66–71
Macabeo-Ong M, Ginzinger DG, Dekker N, McMillan A, Regezi JA, Wong DT, Jordan RC (2002) Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses. Mod Pathol 15:979–987
Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K (1999) Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res 27:4436–4443
Murray AW, Elliott DC, Atkinson MR (1970) Nucleotide biosynthesis from preformed purines in mammalian cells: regulatory mechanisms and biological significance. Prog Nucleic Acid Res Mol Biol 10:87–119
Sirover MA (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta 1432:159–184
Slobin LI (1980) The role of eucaryotic factor Tu in protein synthesis. The measurement of the elongation factor Tu content of rabbit reticulocytes and other mammalian cells by a sensitive radioimmunoassay. Eur J Biochem 110:555–563
Specht K, Richter T, Muller U, Walch A, Werner M, Hofler H (2001) Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol 158:419–429
Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161:1961–1971
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
Walch A, Specht K, Smida J, Aubele M, Zitzelsberger H, Hofler H, Werner M (2001) Tissue microdissection techniques in quantitative genome and gene expression analyses. Histochem Cell Biol 115:269–276
Acknowledgments
We thank Julia Schirrmeier for her excellent technical assistance. This study was supported by grants no. GR 1491/3-1 and SFB 621/C6 from the German Research Council (Deutsche Forschungsgemeinschaft).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
von Smolinski, D., Leverkoehne, I., von Samson-Himmelstjerna, G. et al. Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes. Histochem Cell Biol 124, 177–188 (2005). https://doi.org/10.1007/s00418-005-0013-0
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-005-0013-0