title: Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).
abstract: The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: Shende
authors.initials: S
authors.lastname: Ashish
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India. Electronic address: sdbhure@rediffmail.com.
authors.forename: S K
authors.initials: SK
authors.lastname: Bhure
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: Pillai
authors.initials: P
authors.lastname: Harikrishna
authors.affiliation: Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: S S
authors.initials: SS
authors.lastname: Ramteke
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: V H
authors.initials: VH
authors.lastname: Muhammed Kutty
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: N
authors.initials: N
authors.lastname: Shruthi
authors.affiliation: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: G V P P S
authors.initials: GVPPS
authors.lastname: Ravi Kumar
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: Mahawar
authors.initials: M
authors.lastname: Manish
authors.affiliation: Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: S K
authors.initials: SK
authors.lastname: Ghosh
authors.affiliation: Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar, 243122, Bareilly, U.P., India.
authors.forename: Sarkar
authors.initials: S
authors.lastname: Mihir
mesh_terms: D000818:Animals; D002020:Buffaloes / Q000502:physiology*; D015925:Cryopreservation; D005615:Freezing; D020869:Gene Expression Profiling / Q000592:standards / Q000662:veterinary*; D005786:Gene Expression Regulation / Q000502:physiology*; D008297:Male; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012662:Semen Preservation / Q000662:veterinary*; D013094:Spermatozoa / Q000378:metabolism*
keywords: Buffalo; Cryopreservation; Normalization; Reference gene; Sperm; qRT-PCR