title: Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis.
abstract: The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.
authors.affiliation: Department of Gastroenterology, Affiliated Hospital, Zunyi Medical College, Zunyi, Guizhou 563003, China; Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Zhaoping
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Jinhang
authors.initials: J
authors.lastname: Gao
authors.affiliation: Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Xiuhe
authors.initials: X
authors.lastname: Lv
authors.affiliation: Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yang
authors.affiliation: Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Shilei
authors.initials: S
authors.lastname: Wen
authors.affiliation: Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Tong
authors.affiliation: Division of Peptides Related with Human Diseases, State Key Laboratory of Biotherapy of Human Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China; Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Chengwei
authors.initials: C
authors.lastname: Tang
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010179:Pancreas / Q000737:chemistry / Q000000981:diagnostic imaging / Q000378:metabolism* / Q000473:pathology; D010195:Pancreatitis / Q000235:genetics* / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D005819:Genetic Markers; D011506:Proteins