title: Validation of endogenous control genes for gene expression studies on human ocular surface epithelium.
abstract: OBJECTIVE
  To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions.
  
  
  METHODS
  The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software.
  
  
  RESULTS
  The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable.
  
  
  CONCLUSIONS
  This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium.
authors.affiliation: Division of Ophthalmology and Visual Sciences, Queen's Medical Centre, Nottingham, United Kingdom.
authors.forename: Bina
authors.initials: B
authors.lastname: Kulkarni
authors.forename: Imran
authors.initials: I
authors.lastname: Mohammed
authors.forename: Andrew
authors.initials: A
authors.lastname: Hopkinson
authors.forename: Harminder Singh
authors.initials: HS
authors.lastname: Dua
mesh_terms: D000199:Actins / Q000235:genetics; D002102:Cadaver; D003228:Conjunctiva / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D021984:Cyclophilin A / Q000235:genetics; D004848:Epithelium / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D005966:Glucuronidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012269:Ribosomal Proteins / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics
chemical_list: D000199:Actins; D005136:Eye Proteins; D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins; D035181:TATA-Box Binding Protein; D001613:beta 2-Microglobulin; C054675:ribosomal protein P0; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase; D010735:Phosphoglycerate Kinase; D005966:Glucuronidase; D021984:Cyclophilin A