title: The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data.
abstract: BACKGROUND
  Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0).
  
  
  RESULTS
  We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively.
  
  
  CONCLUSIONS
  The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).
authors.affiliation: Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Dragos
authors.initials: D
authors.lastname: Scarlet
authors.affiliation: Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Ertl
authors.affiliation: Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Aurich
authors.affiliation: Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Ralf
authors.initials: R
authors.lastname: Steinborn
mesh_terms: D000818:Animals; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000662:veterinary; D006736:Horses / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010053:Ovary / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods / Q000662:veterinary*; D012984:Software
chemical_list: D012333:RNA, Messenger