5084 MOLECULAR MEDICINE REPORTS 13: 5084-5092, 2016 Quantitative analysis of gene expression in fixed colorectal carcinoma samples as a method for biomarker validation BEATA OSTASIEWICZ1,2*, PAWEŁ OSTASIEWICZ1*, KAMILA DUŚ‑SZACHNIEWICZ1, KATARZYNA OSTASIEWICZ3 and PIOTR ZIÓŁKOWSKI1 1Department of Pathology, Wrocław Medical University, Wrocław 50‑368; 2Central Laboratory, Diagnostyka Sp. z o.o. Medical Laboratories, Wrocław 50‑041; 3Department of Statistics, Wrocław University of Economics, Wrocław 53‑345, Poland Received September 16, 2015; Accepted March 9, 2016 DOI: 10.3892/mmr.2016.5200 Abstract. Biomarkers have been described as the future of carcinogenesis, however the current study provided strong oncology. Modern proteomics provide an invaluable tool for evidence to support their use as biomarkers. Thus, it was the near-whole proteome screening for proteins expressed concluded that combination of RT-qPCR with proteomics differently in neoplastic vs. healthy tissues. However, in order offers a powerful methodology for biomarker identification, to select the most promising biomarkers, an independent which can be used to analyze FFPE samples. method of validation is required. The aim of the current study was to propose a methodology for the validation of Introduction biomarkers. Due to material availability the majority of large scale biomarker studies are performed using formalin‑fixed Neoplastic diseases are the second major cause of mortality in paraffin-embedded (FFPE) tissues, therefore these were modern societies, and colorectal carcinoma is one of the most selected for use in the current study. A total of 10 genes were prevalent types (1). Biomarkers are prevalent and important selected from what have been previously described as the most in modern medicine. They enable earlier detection of cancer promising candidate biomarkers, and the expression levels were and aid in the prediction of prognosis. However, routine use of analyzed with reverse transcription-quantitative polymerase biomarkers remains low (2). chain reaction (RT-qPCR) using calibrator normalized relative Proteomic technology can aid in more rapid progress quantification with the efficiency correction. For 6/10 analyzed in the search for biomarkers. However, despite more than genes, the results were consistent with the proteomic data; for 15 years of proteomic research, no effective novel biomarkers the remaining four genes, the results were inconclusive. The have been identified for colorectal carcinoma, hundreds of upregulation of karyopherin α 2 (KPNA2) and chromosome candidate biomarkers have been identified, however none are segregation 1-like (CSE1L) in colorectal carcinoma, in addition currently used in clinical practice (3,4). A key difficulty is the to downregulation of chloride channel accessory 1 (CLCA1), selection of biomarkers for further investigation; due to the fatty acid binding protein 1 (FABP1), sodium channel, voltage fact that clinical trials for biomarker validation are expen- gated, type VII α subunit (SCN7A) and solute carrier family 26 sive, it is important to select the most promising candidates. (anion exchanger), member 3 (SLC26A3) was confirmed. Unfortunately, in the majority of cases, promising proteins With the combined use of proteomic and genetic tools, it was selected vary between studies, likely due to the differing reported, for the first time to the best of our knowledge, that protocols used. Quantitative data in proteomics are obtained SCN7A was downregulated in colorectal carcinoma at mRNA in relation to external or internal standards, and since the and protein levels. It had been previously suggested that the standards used are different in each experiment, comparing remaining five genes served an important role in colorectal the results is very difficult. Quantitative data has been previ- ously published (5). Being aware of the controversies existing around the label-free proteomics methods (6), it was decided to validate the most promising biomarkers with a method widely recognized as being reliable in quantitative analysis, Correspondence to: Dr Paweł Ostasiewicz, Department the reverse transcription-quantitative polymerase chain reac- of Pathology, Wrocław Medical University, 1 Marcinkowskiego, tion (RT-qPCR) method. Wrocław 50‑368, Poland E-mail: [email protected] Formalin-fixed paraffin-embedded (FFPE) samples are widely available in large numbers, often with clinical *Contributed equally and outcome information attached, making them ideal for biomarker studies. In the current study, routinely processed, Key words: RT-qPCR, differentially expressed genes, FFPE, archival FFPE samples were used. Although it is widely colorectal cancer, biomarkers known that RNA isolated from FFPE tissue is highly degraded (7-11) it has become more commonly accepted that in spite of the poor RNA quality, gene expression OSTASIEWICZ et al: RT-qPCR FOR BIOMARKER VALIDATION USING FFPE SAMPLES 5085 analysis from the fixed tissue is possible. According to the Switzerland) according to the manufacturer's instructions, literature, use of short amplicons and normalization with with certain modifications. Briefly, 100 µl tissue lysis buffer, more than one reference gene increases the accuracy of the 16 µl 10% sodium dodecyl sulfate and 40 µl proteinase K measurements (11,12). It has also been previously identified (20 mg/ml) from the kit were added to each sample, vortexed that this approach can be applied to the analysis of colorectal with a ZX3 Advanced Vortex Mixer (VELP Scientifica SRL, carcinoma (10). Usmate, Italy) and then incubated at 55˚C for 17 h. RNA was pooled by adding two lysates to each High Pure Filter tube. Materials and methods All purification, DNase treatment, subsequent digestion with proteinase K and all other steps were performed according to Human FFPE specimens. Archival FFPE samples of cancer the manufacturer's protocol. RNA was then resuspended in and adjacent normal colon tissue were obtained from the 50 µl Elution buffer. Once the RNA was extracted, it was then Pathology Department of Wrocław Medical University DNase treated to remove any DNA contamination. DNA was (Wrocław, Poland). The study was approved by the ethics eliminated using the RNase-Free DNase Set (Qiagen GmbH, committee of Wrocław Medical University. For the purpose Hilden, Germany) and the Rneasy Mini kit (Qiagen GmbH). of the investigation thirteen blocks containing tumor tissues DNase digestion was performed twice: First in the eluate and and four blocks containing negative surgical margins (used as then on-column. All steps were performed according to the an approximation of healthy tissue) were selected. Detailed manufacturer's instructions. characteristics of the examined patients are provided in The final volume of each sample was 30 µl. The purified Table I. total RNA was stored at ‑80˚C until used for cDNA synthesis. RNA isolation. All work was conducted under conditions that Nucleic acid measurements. The concentration and the purity minimized the exposure to RNases. The bench surface, all the of the RNA were measured with the Picodrop Microliter equipment and the glass slides were cleaned with RNaseZap UV/Vis Spectrophotometer (Picodrop, Ltd., Hinxton, UK). RNase Decontamination solution (Ambion; Thermo Fisher The purity of the RNA was determined by measuring the Scientific, Inc., Waltham, MA, USA) prior to their use, absorbance ratio A260/A280, with the ratio value ~2.0 according to the manufacturer's instructions. Diethyl pyrocar- being accepted as ‘pure’ RNA. The total amount of nucleic bonate (DEPC; Sigma-Aldrich, St. Louis, MO, USA)-treated acids (RNA, DNA) and proteins was assessed by the Qubit water and hematoxylin (0.1%, v/v) were used throughout the 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, histology procedures. Inc., Waltham, MA, USA). The Qubit measurability limit for The FFPE blocks were cut using a standard microtome proteins is 1.0 µg/ml. The measurements of the RNA concen- (Reichert-Jung Hn40; Leica Microsystems GmbH, Wetzlar, tration were conducted using Picodrop and Qubit a minimum Germany) into 10-µm sections, which were then mounted of 2 times for each sample, and the results are presented as the onto glass slides (Superfrost; Menzel-Glaser; Thermo Fisher mean ± standard deviation. Prior to the RT-qPCR reaction, all Scientific, Inc., Braunschweig, Germany). In each case, the first the samples were applied to a concentration of 600 ng/µl. four sections were discarded to exclude any negative effects The total RNA and mRNA integrity, purity and concen- due to exposure to air. In order to avoid any contamination, a tration were assessed by capillary electrophoresis using an new microtome blade was used for each block. Experion Bioanalyzer, Experion RNA HighSens Chips and All sections were incubated on a hotplate at 70˚C for Experion software, version 3.20 (Bio-Rad Laboratories, Inc., 1 min and then were deparaffinized in two changes of xylene Hercules, CA, USA) following the manufacturer's protocol. (Stanlab Sp. J., Lublin, Poland) for 3 and 2 min. Subsequently the samples were rinsed in alcohol (95% solution for 1 min cDNA synthesis. A reverse transcription reaction was and then 70% solution for 90 sec) and finally in DEPC water performed in a 20 µl volume in a PTC-100 Programmable for 90 sec. To identify the tumor-enriched area, sections Thermal Controller (MJ Research, Inc., Quebec, Canada) were stained with DEPC-treated hematoxylin solution using the Transcriptor First Strand cDNA Synthesis