Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a...

Area of Science:

• Molecular Biology
• Genomics
• Livestock Science

Background:

• Quantitative reverse transcription PCR (RT-qPCR) is crucial for gene expression analysis.
• Validating reference genes for RT-qPCR is labor-intensive and requires species-specific primers.
• A need exists for a universal primer set to facilitate inter-species gene expression studies.

Purpose of the Study:

• To design and test a multi-species primer set for validating reference genes.
• To enable inter-genus RT-qPCR gene expression analysis.
• To simplify and improve the accuracy of gene expression studies in livestock.

Main Methods:

• Designed primers for ten housekeeping genes using transcript sequences from various livestock species.
• Tested primer set performance in Bos taurus (cattle), Bubalus bubalis (buffaloes), Capra hircus (goats), and Ovis aries (sheep) cDNA.
• Evaluated primer efficiency and performed RT-qPCR data normalization using multiple algorithms (Bestkeeper, GeNorm, Norm-finder, Delta CT, RefFinder).

Main Results:

• All ten gene transcripts were detected across the tested livestock species.
• Eight reference genes achieved high primer efficiency (95.54-98.39%) in B. taurus-O. aries fibroblast cDNA.
• Inter-genus RT-qPCR analysis revealed significant up-regulation of TLR4 and ZFX gene transcripts in B. taurus fibroblasts.
• In silico analysis predicted broad applicability across at least 114 mammalian species.

Conclusions:

• The developed multi-species primer set effectively enables inter-genus gene expression analysis between Ovis aries and Bos taurus fibroblasts.
• This approach simplifies reference gene validation for RT-qPCR across diverse species.
• The study highlights the potential for broad application in mammalian transcriptomics and reveals species-specific gene expression patterns.