Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

Abstract

Quantitative reverse transcription PCR (RT-qPCR) remains as an accurate approach for gene expression analysis but requires labor-intensive validation of reference genes using species-specific primers. To ease such demand, the aim was to design and test a multi-species primer set to validate reference genes for inter-genus RT-qPCR gene expression analysis. Primers were designed for ten housekeeping genes using transcript sequences of various livestock species. All ten gene transcripts were detected by RT-PCR in Bos taurus (cattle), Bubalus bubalis (buffaloes), Capra hircus (goats), and Ovis aries (sheep) cDNA. Primer efficiency was attained for eight reference genes using B . taurus—O . aries fibroblast cDNA (95.54–98.39%). The RT-qPCR data normalization was carried out for B . taurus vs. O . aries relative gene expression using Bestkeeper, GeNorm, Norm-finder, Delta CT method, and RefFinder algorithms. Validation of inter-genus RT-qPCR showed up-regulation of TLR4 and ZFX gene transcripts in B . taurus fibroblasts, irrespectively of normalization conditions (two, three, or four reference genes). In silico search in mammalian transcriptomes showed that the multi-species primer set is expected to amplify transcripts of at least two distinct loci in 114 species, and 79 species would be covered by six or more primers. Hence, a multi-species primer set allows for inter-genus gene expression analysis between O . aries and B . taurus fibroblasts and further reveals species-specific gene transcript abundance of key transcription factors.

Housekeeping genes for RT-qPCR in ovine preimplantation embryos

“…Primer design was based on the following criteria: amplicon size (70-200 bp), GC content (40-60%), primer size (19-23 bases), and avoiding primer dimers and hairpin loops (Table 1). Primer sequences were also obtained from previous reports (Moura et al, 2018(Moura et al, , 2019Silva et al, 2018).…”

“…Among the available tools for gene expression analysis, quantitative reverse transcription PCR (RT-qPCR) is widely used for accurate measurements of both relative and absolute gene transcript abundance (VanGuilder et al, 2008;Bustin, 2010;Moura et al, 2019), even for loci of low transcript abundance (Derveaux et al, 2010). The RT-qPCR combines accuracy, specificity and reproducibility (Luchsinger et al, 2014).…”