Open AccessInter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set
Author(s) -
Marcelo Tigre Moura,
Roberta L. O. Silva,
Pábola Santos Nascimento,
José Carlos Ferreira-Silva,
Ludymila Furtado Cantanhêde,
Éderson Akio Kido,
Ana Maria BenkoIseppon,
Marcos Antônio Lemos Oliveira
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0221170
Subject(s) - biology , reference genes , housekeeping gene , gene , gene expression , primer (cosmetics) , capra hircus , genetics , gene expression profiling , complementary dna , in silico , microbiology and biotechnology , zoology , chemistry , organic chemistry
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.