PMID- 31935552 OWN - NLM STAT- MEDLINE DCOM- 20201120 LR - 20201120 IS - 1095-9947 (Electronic) IS - 1050-4648 (Linking) VI - 98 DP - 2020 Mar TI - Transcriptome-based evaluation and validation of suitable housekeeping gene for quantification real-time PCR under specific experiment condition in teleost fishes. PG - 218-223 LID - S1050-4648(20)30018-8 [pii] LID - 10.1016/j.fsi.2020.01.018 [doi] AB - Quantification real-time PCR (qRT-PCR) is a common method in analysis of gene expression, but the stable reference genes for the normalization analysis have not been appreciated before identifying expression pattern of genes in teleost fishes. In this study, we selected eight candidate reference genes (18S, Actin, EF-1alpha, 40S, B2M, TUBA, UBCE and GAPDH) basing on transcriptome analysis and the traditional housekeeping genes, and analyzed the stability of the reference genes in spleen, head kidney and head kidney leukocytes (HKL) after pathogen challenge in Schizothorax prenanti (S. prenanti). Three common programs (geNorm, NormFinder and Bestkeeper) were used to evaluate the stability of the candidate reference genes. Two reference genes, Actin and EF-1alpha presented higher stability, while 18S and GAPDH were the lower stable genes, both in in vitro and in vivo. An important immune gene, toll-like receptor 22a (TLR22a), was selected to validate the stability of the proposed reference genes (Actin and EF-1alpha) across different experiment treatments. The results reveal that Actin and EF-1alpha are quite suitable reference genes for the normalization analysis. Otherwise, using the most stable gene Actin to validate the reliable of transcriptome data showed the high correlation between the fold change of transcriptome data and qRT-PCR data. In conclusion, our study not only acquired the suitable reference gene for the qRT-PCR assay under specific experiment condition, but also provided a comprehensive method to evaluate and validate the reference gene based on transcriptome analysis in teleost fishes. CI - Copyright (c) 2020 Elsevier Ltd. All rights reserved. FAU - Li, Yunkun AU - Li Y AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Han, Jiabei AU - Han J AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Wu, Jiayu AU - Wu J AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Li, Dong AU - Li D AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Yang, Xixi AU - Yang X AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Huang, Anqi AU - Huang A AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Bu, Guixian AU - Bu G AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Meng, Fengyan AU - Meng F AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Kong, Fanli AU - Kong F AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Cao, Xiaohan AU - Cao X AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Han, Xingfa AU - Han X AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. FAU - Pan, Xiaofu AU - Pan X AD - State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, PR China. FAU - Yang, Shiyong AU - Yang S AD - Department of Aquaculture, Sichuan Agricultural University, 625014, Sichuan, PR China. FAU - Zeng, Xianyin AU - Zeng X AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. Electronic address: xyzeng@sicau.edu.cn. FAU - Du, Xiaogang AU - Du X AD - Department of Engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Ya'an, 625014, Sichuan, PR China. Electronic address: duxiaogang@sicau.edu.cn. LA - eng PT - Journal Article DEP - 20200111 PL - England TA - Fish Shellfish Immunol JT - Fish & shellfish immunology JID - 9505220 RN - 0 (Actins) RN - 0 (Fish Proteins) RN - 0 (Peptide Elongation Factor 1) RN - 0 (Toll-Like Receptors) SB - IM MH - Actins/genetics MH - Animals MH - Cyprinidae/*genetics MH - Fish Proteins/genetics MH - Gene Expression Profiling/*standards MH - *Genes, Essential MH - Peptide Elongation Factor 1/genetics MH - Real-Time Polymerase Chain Reaction/*standards MH - Reference Standards MH - Reproducibility of Results MH - Toll-Like Receptors/genetics MH - Transcriptome OTO - NOTNLM OT - Normalization OT - Reference genes OT - Stability OT - Teleost fishes OT - Transcriptome analysis OT - qRT-PCR EDAT- 2020/01/15 06:00 MHDA- 2020/11/21 06:00 CRDT- 2020/01/15 06:00 PHST- 2019/11/13 00:00 [received] PHST- 2020/01/04 00:00 [revised] PHST- 2020/01/10 00:00 [accepted] PHST- 2020/01/15 06:00 [pubmed] PHST- 2020/11/21 06:00 [medline] PHST- 2020/01/15 06:00 [entrez] AID - S1050-4648(20)30018-8 [pii] AID - 10.1016/j.fsi.2020.01.018 [doi] PST - ppublish SO - Fish Shellfish Immunol. 2020 Mar;98:218-223. doi: 10.1016/j.fsi.2020.01.018. Epub 2020 Jan 11.