Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set.
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 referenc...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2019-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0221170 |
| _version_ | 1818682301895147520 |
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| author | Marcelo T Moura Roberta L O Silva Pábola S Nascimento José C Ferreira-Silva Ludymila F Cantanhêde Ederson A Kido Ana M Benko-Iseppon Marcos A L Oliveira |
| author_facet | Marcelo T Moura Roberta L O Silva Pábola S Nascimento José C Ferreira-Silva Ludymila F Cantanhêde Ederson A Kido Ana M Benko-Iseppon Marcos A L Oliveira |
| author_sort | Marcelo T Moura |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T10:16:41Z |
| format | Article |
| id | doaj.art-d92504310a6b40189a9a02d0dfaf4eab |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T10:16:41Z |
| publishDate | 2019-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d92504310a6b40189a9a02d0dfaf4eab2022-12-21T21:52:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032019-01-01148e022117010.1371/journal.pone.0221170Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set.Marcelo T MouraRoberta L O SilvaPábola S NascimentoJosé C Ferreira-SilvaLudymila F CantanhêdeEderson A KidoAna M Benko-IsepponMarcos A L OliveiraQuantitative 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.https://doi.org/10.1371/journal.pone.0221170 |
| spellingShingle | Marcelo T Moura Roberta L O Silva Pábola S Nascimento José C Ferreira-Silva Ludymila F Cantanhêde Ederson A Kido Ana M Benko-Iseppon Marcos A L Oliveira Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. PLoS ONE |
| title | Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. |
| title_full | Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. |
| title_fullStr | Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. |
| title_full_unstemmed | Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. |
| title_short | Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set. |
| title_sort | inter genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi species primer set |
| url | https://doi.org/10.1371/journal.pone.0221170 |
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