title: Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model.
abstract: BACKGROUND
  With the development of real-time quantitative polymerase chain reaction (RT-qPCR) and intensive research on acute graft-versus-host disease (GVHD), selecting the best reference gene for normalization of RT-qPCR analysis in a GVHD model becomes more and more important. In this study, we aimed to identify suitable reference genes for mRNA studies in an intestinal GVHD mouse model after bone marrow transplantation (BMT).
  
  
  METHODS
  BALB/c recipients received 7.5 Gy total body irradiation (TBI) followed by injection of 5 × 10(6) bone marrow cells, without infusion of spleen cells for BMT, with infusion of 5 × 10(5) or 2.5 × 10(6) spleen cells for mild or moderate GVHD, respectively. Healthy mice were chosen as normal control subjects. Duodenum, jejunum, ileum, colon, and small intestine were collected at days 7, 14, 21, and 28 after transplantation. Transcription levels of 9 candidate genes, B2M, SDHA, HPRT, ACTB, GAPDH, HMBS, TBP, YWHAZ, and RPLP0, in each tissue were measured with the use of RT-qPCR. Combined data from these tissues in each group were defined as all samples. The expression stability of these genes was analyzed with the use of Genorm, Normfinder, Bestkeeper, and ΔCt.
  
  
  RESULTS
  Our results showed that in all samples, ACTB and HMBS displayed the highest and lowest expression levels, respectively. Genorm identified HRPT and SDHA as the most stable reference genes, whereas Normfinder and ΔCt method showed HPRT as the most stably expressed gene. Bestkeeper ranked YWHAZ and HPRT as the top 2 most suitable genes. In conclusion, HPRT was recommended as the most suitable reference gene after comprehensive ranking, suggesting that it could be used as an internal control for mRNA studies in intestinal GVHD after BMT.
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China; Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: X
authors: X
authors: Li
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China; Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: J
authors: J
authors: Qiao
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: N
authors: N
authors: Yang
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: H
authors: H
authors: Mi
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: P
authors: P
authors: Chu
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: Y
authors: Y
authors: Xia
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: H
authors: H
authors: Yao
authors: Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: Y
authors: Y
authors: Liu
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: K
authors: K
authors: Qi
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China.
authors: Z
authors: Z
authors: Yan
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China; Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China. Electronic address: zengly2000@163.com.
authors: L
authors: L
authors: Zeng
authors: Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China; Key Laboratory of Bone Marrow Stem Cells, Xuzhou, Jiangsu Province, People's Republic of China; Blood Diseases Institute, Xuzhou Medical College, Xuzhou, People's Republic of China. Electronic address: lihmd@163.com.
authors: K
authors: K
authors: Xu
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006086:Graft vs Host Disease / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007421:Intestine, Small / Q000637:transplantation*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012333:RNA, Messenger