effect of embryonic genotype on reference gene selection for RT-qPCR normalization.

Area of Science:

• Animal Science
• Molecular Biology
• Genetics

Background:

• Accurate gene expression analysis in rabbit embryos requires appropriate internal control genes for data normalization.
• Previous studies identified stable reference genes in oocytes and pre-implantation embryos, but the impact of embryonic genotype remains unclear.

Purpose of the Study:

• To evaluate the mRNA transcription stability of histone (H2afz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in rabbit blastocysts from lines selected for litter size and weight gain.
• To assess the influence of embryonic genotype on gene expression analysis using these reference genes and specific embryonic transcripts.

Main Methods:

• Utilized geNorm, NormFinder, and BestKeeper software to analyze the stability of H2afz and GAPDH mRNA levels.
• Examined the expression of embryonic transcripts including POU5F1, erbB3, TGF-beta2, VEGF, and Ifn-gamma.

Main Results:

• H2afz and GAPDH demonstrated high stability in rabbit blastocysts selected for litter size.
• Embryonic genotype significantly affected target gene expression when only one reference gene was used.
• GAPDH proved to be a more reliable reference gene than H2afz across both studied embryo genotypes.

Conclusions:

• A normalization factor combining H2afz and GAPDH is recommended for RT-qPCR in rabbit embryos of varying genotypes.
• GAPDH is the preferred single reference gene for gene expression studies in these rabbit embryo models.