title: Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density.
abstract: BACKGROUND
  Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions.
  
  
  METHODS
  During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard.
  
  
  RESULTS
  Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed.
  
  
  CONCLUSIONS
  The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.
authors.forename: Vijay S
authors.initials: VS
authors.lastname: Baddela
authors.forename: Anja
authors.initials: A
authors.lastname: Baufeld
authors.forename: Vengala R
authors.initials: VR
authors.lastname: Yenuganti
authors.affiliation: Animal Biochemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India. vanselow@fbn-dummerstorf.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Vanselow
authors.forename: Dheer
authors.initials: D
authors.lastname: Singh
mesh_terms: D000818:Animals; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D020043:Genes, Essential / Q000235:genetics; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D035181:TATA-Box Binding Protein / Q000235:genetics; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D001613:beta 2-Microglobulin / Q000235:genetics
chemical_list: D035181:TATA-Box Binding Protein; D001613:beta 2-Microglobulin; D005640:Follicle Stimulating Hormone