title: Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging.
abstract: The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P>0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P=0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.
authors.affiliation: Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Muhammad Irfan-Ur-Rehman
authors.initials: MI
authors.lastname: Khan
authors.affiliation: Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Fernanda Caminha Faustino
authors.initials: FC
authors.lastname: Dias
authors.affiliation: Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, QC, G1V 0A6, Canada.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Dufort
authors.affiliation: Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Vikram
authors.initials: V
authors.lastname: Misra
authors.affiliation: Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, QC, G1V 0A6, Canada.
authors.forename: Marc-Andre
authors.initials: MA
authors.lastname: Sirard
authors.affiliation: Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Jaswant
authors.initials: J
authors.lastname: Singh
mesh_terms: D000375:Aging; D000465:Algorithms; D000818:Animals; D017849:Animals, Inbred Strains; D002417:Cattle; D020717:Eukaryotic Initiation Factor-2B / Q000235:genetics / Q000378:metabolism; D005260:Female; D005300:Fertility Agents, Female / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000378:metabolism* / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009866:Oogenesis / Q000187:drug effects; D006080:Ovarian Follicle / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010060:Ovulation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D000072260:RNA Splicing Factors / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics / Q000378:metabolism; D012525:Saskatchewan; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D020717:Eukaryotic Initiation Factor-2B; D005300:Fertility Agents, Female; D020033:Protein Isoforms; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D017413:Ribonucleoprotein, U2 Small Nuclear; C088049:splicing factor 3a; D005640:Follicle Stimulating Hormone; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases