Methodology article

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

Ann-Britt Nygard, Claus B Jørgensen, Susanna Cirera and Merete Fredholm^*

• * Corresponding author: Merete Fredholm mf@life.ku.dk

Author Affiliations

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

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BMC Molecular Biology 2007, 8:67 doi:10.1186/1471-2199-8-67

Published: 15 August 2007

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.

Conclusion

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.