Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes

Abstract With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ , RPL-13A , PGK1, B2M , GAPDH , HPRT , SDHA and ACTB . Using established algorithms for reference gene normalization “geNorm” and “Norm Finder”, PGK1 , GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1 , YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep.