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The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data.

Scarlet D, Ertl R, Aurich C, Steinborn R - PLoS ONE (2015)

Bottom Line: The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M).The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary.In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

View Article: PubMed Central - PubMed

Affiliation: Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine, Vienna, Austria.

ABSTRACT

Background: Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0).

Results: We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively.

Conclusions: The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

No MeSH data available.


Expression uniformity analysis using the RefFinder software.The individual genes or the normalization factor calculated from the best-ranking, not co-expressed pair of genes are depicted. Only the “expressors” of highest and lowest ranks are presented.
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pone.0142122.g003: Expression uniformity analysis using the RefFinder software.The individual genes or the normalization factor calculated from the best-ranking, not co-expressed pair of genes are depicted. Only the “expressors” of highest and lowest ranks are presented.

Mentions: Expression stability rankings produced by analytic algorithms like NormFinder and BestKeeper are often in overall agreement [27]. However, a discrepancy in stability rankings and outcomes between different analytical methods has been reported [24,25,48]. To increase the chance for deducing a “consensus” in the expression uniformity ranking of the novel candidate reference genes we used the comprehensive tool RefFinder (S5 File). The comprehensive approach identified SNRPD3—a core component of the spliceosome and VCP—an ATPase of the endoplasmic reticulum that prevents mutations caused by DNA damage, as the most stably expressed pair of genes in the biological context studied. Traditional RGs like GAPDH, B2M, ACTB and UBB were less uniformly expressed as documented by their ranks 3 and 12 to 14, respectively. If normalization should be performed with more than one RG, the pair SNRPD3 and VCP is recommended for calculating a normalization factor (Fig 3).


The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data.

Scarlet D, Ertl R, Aurich C, Steinborn R - PLoS ONE (2015)

Expression uniformity analysis using the RefFinder software.The individual genes or the normalization factor calculated from the best-ranking, not co-expressed pair of genes are depicted. Only the “expressors” of highest and lowest ranks are presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4633174&req=5

pone.0142122.g003: Expression uniformity analysis using the RefFinder software.The individual genes or the normalization factor calculated from the best-ranking, not co-expressed pair of genes are depicted. Only the “expressors” of highest and lowest ranks are presented.
Mentions: Expression stability rankings produced by analytic algorithms like NormFinder and BestKeeper are often in overall agreement [27]. However, a discrepancy in stability rankings and outcomes between different analytical methods has been reported [24,25,48]. To increase the chance for deducing a “consensus” in the expression uniformity ranking of the novel candidate reference genes we used the comprehensive tool RefFinder (S5 File). The comprehensive approach identified SNRPD3—a core component of the spliceosome and VCP—an ATPase of the endoplasmic reticulum that prevents mutations caused by DNA damage, as the most stably expressed pair of genes in the biological context studied. Traditional RGs like GAPDH, B2M, ACTB and UBB were less uniformly expressed as documented by their ranks 3 and 12 to 14, respectively. If normalization should be performed with more than one RG, the pair SNRPD3 and VCP is recommended for calculating a normalization factor (Fig 3).

Bottom Line: The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M).The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary.In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

View Article: PubMed Central - PubMed

Affiliation: Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine, Vienna, Austria.

ABSTRACT

Background: Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0).

Results: We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively.

Conclusions: The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

No MeSH data available.