Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes

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.

Development and evaluation of canine reference genes for accurate quantification of gene expression

“…3, the amplicon size of ACTB is approximately double the size of the other amplicons tested. More important is the observation by von Smolinski and coworkers [17], who found equal G3P and ACTB qPCR expression levels even in formalin-fixed and paraffin-embedded canine liver samples. This contradiction with our results can be caused by the fact that their primers resulted in an ACTB amplicon within the size range of the other reference genes tested by us.…”

“…In human biomedical research, there are several reports on evaluating reference genes [9][10][11] or the qPCR technique [12][13][14]. To our knowledge, the evaluation of reference genes for accurate normalization of gene expression has not been done extensively for domestic animals [15][16][17].…”

Gene expression analysis using long-term preserved formalin-fixed and paraffin-embedded tissue of non-small cell lung cancer

“…The majority of studies include fresh tissues or cells which are formalinfixed and paraffin-embedded at the time of the study, in order to mimic a fixation process (1,(3)(4)(7)(8)(9)(10)(11)(12). However, FFPE tissues in histopathology departments have in general been stored for several years and since storage time may affect downstream applications, it seems more relevant to use stored tissues to evaluate the methods for nucleic acid extraction and gene expression analysis.…”

“…Several studies use only a few control genes (1,3,7,8,12) and the selection of genes for qPCR has been discussed (12). Microarray analysis is the technique of choice to analyze a broader spectrum of genes, but we found only a few studies evaluating the performance of FFPE RNA in microarray analysis.…”

Techniques for maximizing the performance of molecular pathology testing: responsibilities of all pathologists

“…The advantage of liquid-based studies is that they can be easily applied and put into protective fluid after obtaining the sample, which is necessary for the protection of the nucleic acid, immediately. Furthermore, protective liquids used in liquid-based studies do not cause the problem of DNA degradation in formalin-fixed tissues (92).…”