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Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

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Abstract

In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

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Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant no. 22590642).

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Authors and Affiliations

  1. Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585, Osaka, Japan

    Qi Wang, Takaki Ishikawa, Tomomi Michiue & Hitoshi Maeda

  2. Medico-legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan

    Qi Wang, Takaki Ishikawa, Tomomi Michiue & Hitoshi Maeda

  3. Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China

    Bao-Li Zhu & Da-Wei Guan

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  1. Qi Wang
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  2. Takaki Ishikawa
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Correspondence to Qi Wang.

Electronic supplementary material

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Supplementary Material 1

Introduction of candidate reference genes (n = 32). “_s” suffix indicates the assay’s primers and probes are designed within a single exon and will detect genomic DNA. “_m” indicates the assay’s probe spans an exon junction and will not detect genomic DNA. “_g” indicates the assay may detect genomic DNA. The “context sequence” is the nucleotide sequence surrounding the region to which the probe binds. Primer and probe sequences for TaqMan assays are not available. Detailed information for each TaqMan Assay is available from Applied Biosystems. (PPT 121 kb)

Supplementary Material 2

Linear regression between RIN and relative non-normalized quantities of candidate reference genes. Genes with significant correlations with RIN are shown in bold. RIN, RNA integrity number; R 2, coefficients of determination; b0, the intercept of the regression line with the y-axis; b1, the slope of the regression line. (PPT 114 kb)

Supplementary Material 3

Pearson correlation coefficients (R 2) between relative non-normalized quantities of each pair of candidate reference genes. (XLS 52 kb)

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Wang, Q., Ishikawa, T., Michiue, T. et al. Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper. Int J Legal Med 126, 943–952 (2012). https://doi.org/10.1007/s00414-012-0774-7

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