Methodology article

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

Ewart W Kuijk¹, Leonie du Puy^1,2, Helena TA van Tol¹, Henk P Haagsman², Ben Colenbrander¹ and Bernard AJ Roelen^1*

• * Corresponding author: Bernard AJ Roelen b.a.j.roelen@vet.uu.nl

Author Affiliations

¹ Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands

² Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

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BMC Developmental Biology 2007, 7:58 doi:10.1186/1471-213X-7-58

Published: 31 May 2007

Abstract

Background

In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.

Results

In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.

Conclusion

Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.