title: Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR.
abstract: BACKGROUND
  Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR.
  
  
  RESULTS
  In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH.
  
  
  CONCLUSIONS
  Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.
authors: University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences, Division of Genetics and Bioinformatics, Groennegaardsvej 3, 1870 Frederiksberg C, Denmark. abn@life.ku.dk
authors: Ann-Britt
authors: AB
authors: Nygard
authors: Claus B
authors: CB
authors: Jørgensen
authors: Susanna
authors: S
authors: Cirera
authors: Merete
authors: M
authors: Fredholm
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D015870:Gene Expression; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012313:RNA / Q000378:metabolism; D012016:Reference Values; D034421:Sus scrofa / Q000033:anatomy & histology / Q000235:genetics* / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D012313:RNA
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