151 EVALUATION OF REFERENCE GENE TRANSCRIPT STABILITY DURING EARLY PRE-IMPLANTATION DEVELOPMENT AND IN SPERMATOGONIAL CELLS IN THE CAT

Abstract

Differences in the stability of commonly used reference genes have been discovered between species and among different tissue types. These inconsistencies underscore the importance of validation and selection of an appropriate reference for normalization of gene expression to an endogenous control in RT-qPCR experiments. Three different reference transcripts (GAPDH, RPS19 and 18S rRNA) of 3 different functional categories were selected for evaluation during pre-implantation embryonic development and in spermatogonial cells in the domestic cat. Amplification efficiency was done by standard curve analysis with 5-fold template dilutions. For pre-implantation analysis, transcripts were assayed at 4 developmental stages: 2- to 6-cell (2–6C), 8- to 16-cell (8–16C), morula (M) and blastocyst (BL). Embryos were from pools of 4 to 10 in vitro matured and fertilized domestic cat embryos. For spermatogonia, isolates of 20 000 to 30 000 cells were assayed from single testis isolates following a collagenase and trypsin with DNase digestion and Percoll gradient separation. Total mRNA was isolated using the Cells-to-cDNA II Kit, with a minimum of 2 biological replicates for each sample type. The RNA quantitation was done by RiboGreen analysis and 47 ng of RNA was used for cDNA synthesis. Transcript abundance was detected in 2 technical replicates per sample by SYBR Green chemistry and data were analysed with NormFinder and one-way ANOVA. Each transcript showed varying levels of expression throughout development and among embryonic stages and in spermatogonial cells. The GADPH and 18S rRNA transcripts were expressed at higher levels in BL than in 2–6C and 8–16C, respectively. Contrarily, transcript levels of RSP19 were lower in BL compared with levels at 2–6C. Even though differences were observed among embryonic stages, analysis with NormFinder indicated that the most stable reference gene throughout early pre-implantation development was RPS19. When each stage was analysed separately, 18S rRNA was the most stable at 2–6C, whereas RPS19 was found to be the most stable at 8–16C, M and BL stages. In spermatogonial samples, GAPDH was the most stably expressed gene. Our results support the selection of an appropriate reference gene based on the needs of the experimental design. We conclude from these findings that when gene expression throughout the duration of early embryonic development is examined, RPS19 is the preferred selection. If gene expression is analysed within discrete time points of development only, it is appropriate to select 18S rRNA at the 2–6C stage and RPS19 for 8–16C, M and BL stage embryos. For spermatogonial samples, if comparing only different biological replicates, GAPDH should be selected for normalization of expression; however if the purpose of the experiment is to assay genes that are also expressed in BL, the most stably expressed reference gene is RPS19.