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Stability of Endogenous Reference Genes in Postmortem Human Brains

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Stability of Endogenous Reference Genes in Postmortem Human Brains Int J Legal Med DOI 10.1007/s00414-012-0774-7 ORIGINAL ARTICLE Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper Qi Wang & Takaki Ishikawa & Tomomi Michiue & Bao-Li Zhu & Da-Wei Guan & Hitoshi Maeda Received: 10 July 2012 /Accepted: 14 September 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract In forensic molecular pathology, quantitative met standard efficiency criteria. Validation of their stability real-time polymerase chain reaction (RT-qPCR) provides and suitability as reference genes using geNorm suggested a rapid and sensitive method to investigate functional IPO8 and POLR2A as the most stable ones, and NormFinder changes in the death process. Accurate and reliable indicated that IPO8 and POP4 had the highest expression relative RT-qPCR requires ideal amplification efficien- stabilities, while BestKeeper highlighted ABL1 and cies of target and reference genes. However, the ampli- ELF1 as reference genes with the least overall variation. fication efficiency, changing during PCR, may be Combining these three algorithms suggested the genes overestimated by the traditional standard curve method. IPO8, POLR2A, and PES1 as stable endogenous refer- No single gene meets the criteria of an ideal endoge- ences in RT-qPCR analysis of human brain samples, nous reference. Therefore, it is necessary to select suitable with YWHAZ, PPIA, HPRT1, and TBP being the least reference genes for specific requirements. The present stable ones. These findings are inconsistent with those of study evaluated 32 potential reference genes in the human previous studies. Moreover, the relative stability of target and brain of 15 forensic autopsy cases using three different statis- reference genes remains unknown. These observations sug- tical algorithms, geNorm, NormFinder, and BestKeeper. On gest that suitable reference genes should be selected on the RT-qPCR data analyses using a completely objective and basis of specific requirements, experiment conditions, and the noise-resistant algorithm (Real-time PCR Miner), 24 genes characteristics of target genes in practical applications. Keywords Quantitative real-time PCR . Endogenous reference genes . geNorm . NormFinder . BestKeeper . Electronic supplementary material The online version of this article Human postmortem brain material (doi:10.1007/s00414-012-0774-7) contains supplementary material, which is available to authorized users. Q. Wang (*) : T. Ishikawa : T. Michiue : H. Maeda Department of Legal Medicine, Introduction Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan Quantitative real-time reverse transcriptase polymerase e-mail: [email protected] chain reaction (RT-qPCR) is a sensitive, efficient, and : : : reliable molecular technique to determine changes in Q. Wang T. Ishikawa T. Michiue H. Maeda mRNA expressions [1, 2]andhasalsobeenusedin Medico-legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), the field of forensic sciences, including DNA technolo- Osaka, Japan gy for the criminal justice process as well as postmor- : tem gene expression analysis [3, 4]. RT-qPCR can B.-L. Zhu D.-W. Guan detect the systemic pathophysiological changes involved Department of Forensic Pathology, China Medical University School of Forensic Medicine, in the death process that cannot be detected by morphol- Shenyang, China ogy [5–8]. This procedure may be useful in investigating Int J Legal Med functional alterations in the brain after insults in forensic Material and methods neuropathology [9]. The most common procedures in RT-qPCR are rela- Sample collection tive measurements of gene expressions of interest after normalization using endogenous reference gene(s). Ac- Human brains of forensic autopsy cases (n015) at our curate and reliable relative RT-qPCR requires ideal am- institute, including blunt brain injury (n012) and sudden plification efficiencies of target and reference genes cardiac death (SCD, n03), were examined. A thorough [10]; selection of adequate reference genes is essential. neuropathological analysis was performed as part of our Previous studies suggested successful application of routine investigation and cases with any preexisting neuro- conventional reference genes for the myocardium, lungs, logical pathologies were excluded in the present study. SCD kidneys, and skeletal muscle [7, 11–14]. For estimating cases included those due to acute ischemic heart disease the amplification efficiency, however, conventional stan- with or without apparent focal myocardial necrosis dard curve method is time consuming, requiring the (infarction) without any evidence of cause of death other production of repeatable and reliable standards [15, than a cardiac attack [31]. Details are shown in Table 1. 16]. Furthermore, the amplification efficiency changes Postmortem interval was defined as the estimated time from during PCR, being relatively stable in the early expo- death to autopsy and survival time was the estimated period nential phase and gradually declining in later cycles [1, from the onset of fatal insult to death; these were estimated 17]; it may be overestimated by the standard curve on the basis of autopsy findings and circumstantial evidence method [18]. There are several alternative methods for in autopsy documents. Sample collections and analyses calculating the amplification efficiency on the basis of described below were performed within the framework of raw data collected during PCR [17–24], which has been our routine casework, following the Autopsy Guidelines reported to be more accurate than that derived from the stan- (2009) and Ethical Guidelines (1997 and 2003) of the Jap- dard curve method [1, 18, 25]. Furthermore, expressions of anese Society of Legal Medicine, approved by our Institu- several conventional housekeeping genes have been shown to tional Ethics Committee. vary due to nutritional or hormonal factors, biological pro- Brain tissue samples were taken from consistent sites in cesses, and/or tissue or cell types; a single housekeeping gene the parietal lobe of left cerebral hemispheres at autopsy as may not meet the criteria of an ideal reference gene [26]. The part of our routine work. In blunt brain injury cases, tissue influence of prolonged agony on RT-qPCR data of the post- mortem human brain has also been reported [27]. Since 2002, several statistical algorithms, such as Table 1 Case profiles (n015) geNorm [28], NormFinder [29], and BestKeeper [30], have been developed to select stably expressed reference Case Age Gender ST PMI A260/A280 RIN genes, and the use of multiple reference genes for number (years) (hours) (hours) ratio accurate normalization in RT-qPCR was proposed. GeN- Blunt brain injury orm determines the expression stability of candidate Case 1 29 F 0.1 13 1.98 5.5 reference genes by gene stability measure (M), selecting Case 2 54 F 2.5 20 1.86 5.1 an optimal number of reference genes out of a larger group Case 3 31 M 3 11 1.97 5.2 of candidate genes. NormFinder evaluates the expression Case 4 44 M 6 26 1.99 5.5 stability of each single reference gene and takes into Case 5 57 M 6 15 1.94 6.4 account intra- and intergroup variations for normaliza- Case 6 35 M 12 22 1.86 5.0 tion. BestKeeper analyzes variabilities in the expression of Case 7 53 M 20 43 1.87 5.7 candidate reference genes by calculation of cycle threshold Case 8 67 M 36 33 1.93 5.7 (Ct) data variations. Case 9 78 M 72 30 1.93 4.5 In the present study, amplification efficiencies in RT- Case 10 59 M 192 27 1.96 4.2 qPCR were calculated for 32 potential reference genes Case 11 65 M 312 34 1.96 4.3 in postmortem human brain tissues using a completely Case 12 79 M 384 31 1.81 1.4 objective and noise-resistant algorithm (Real-time PCR SCD Miner) [24] to select candidate reference genes; then, Case 13 62 F <0.5 14 2.03 6.3 the stability and suitability in RT-qPCR were evaluated Case 14 72 M <0.5 22 1.96 6.9 using three different statistical algorithms, geNorm, NormFinder, and BestKeeper, to examine the efficacy Case 15 16 M <0.5 20 2.00 6.7 of candidates for possible practical application in post- ST survival time, PMI postmortem interval, RIN RNA integrity num- mortem investigations. ber, F female, M male, SCD sudden cardiac death Int J Legal Med specimens distant from primary lesions were selected [32]. Statistical analysis All samples were immediately submerged in 1 ml of RNA stabilization solution (RNAlaterTM, Ambion, Austin) and Amplification efficiencies were calculated from raw fluores- stored at −80 °C until use. cent data (Rn values), using the Real-time PCR Miner program [24]; the arithmetic mean values of amplification Extraction of total RNA and cDNA synthesis efficiencies were used for further relative quantification. Correlation analyses between pairs of parameters were per- Total RNA was isolated from 100 mg of sample using formed arbitrarily using linear regression (Pearson correla- ISOGEN (Nippon Gene, Toyama) according to the ISO- tion) with XLSTAT 2012 (Addinsoft, Paris, France). GEN RNA Extraction Protocol Procedure provided by the To compare gene expression stability and rank, geNorm manufacturer. After extraction, the RNA concentration was [28], NormFinder [29], and BestKeeper [30] algorithms estimated by spectrophotometric analysis using NanoDrop were used. For geNorm, raw Ct values were transformed 1000 (Thermo Scientific, Wilmington, USA). cDNA copies to relative non-normalized quantities (Q), according to geN- ∼ of total RNA were obtained using a High Capacity RNA-to- orm manual (http://medgen.ugent.be/ jvdesomp/genorm/ cDNA kit (Applied Biosystems Japan, Ltd.) then were ad- geNorm_manual.pdf), with regard to the specific amplifica- justed to a concentration equivalent to 5 ng/μl of total RNA tion efficiency calculated by Real-time PCR Miner program, 0 ΔCt using nuclease-free water. by the equation Q E , where E is the exponential ampli- fication and ΔCt0min Ct−sample Ct, where min Ct is the lowest Ct value of each gene and sample Ct is the Ct value Evaluation of the quality and integrity of RNA samples of the sample being transformed [34, 35].
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