Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach

Selvarajah, Gayathri Thevi; Bonestroo, Floor A. S.; Sprang, E.P.M.; Kirpensteijn, Jolle; Mol, Jan A.

doi:10.1186/s12917-017-1281-3

Cited by 22 publications

(18 citation statements)

References 36 publications

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“…The RefFinder software comprehensively ranks the candidate reference genes based on the geometric mean (GM) values of the results from the four different statistical algorithms described above ( Xie et al, 2012 ). With the help of these five statistical algorithms, much research on the validation of reference genes under different experimental conditions has been reported ( Liu et al, 2017 ; Sadangi et al, 2017 ; Sprang et al, 2017 ; Mughal et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Zhou

Chen

et al. 2018

Front. Genet.

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Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

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Section: Introductionmentioning

confidence: 99%

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Zhou

Chen

et al. 2018

Front. Genet.

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“…In this study, 12 traditional reference genes were selected as candidate reference genes after literature search [3][4][5][6][7], then subjected to transcriptome analysis. These 12 relatively stable candidate reference genes in polyploid of Cyprinus carpio and Carassius auratus were evaluated using BestKeeper, NormFinder and geNorm, three reference gene stability analysis softwares that are widely used in the reference gene selection in species such as animals, micro-organisms and plants [45,[47][48][49][50]. For the polyploid tissues, the top three stable reference genes and RPS18, those by NormFinder were RPS18, RPS5 and RPLP2, and those by geNorm were RPS5, RPS18 and RPL7.…”

Section: Discussionmentioning

confidence: 99%

“…Our data showed that the expression levels of RPS5 and RPS18 were relatively stable not only in the polyploid fish tissues in vivo but also in the cultured cells in vitro. For some reasons, such as extreme conservativation of evolution, highly and stably expression in different types of cells and tissues, several components of ribosomes were selected as reference genes [47,[51][52][53][54][55]. In eukaryotic cells, the ribosomal RNA (rRNA) genes exist in multiple copies, however, without poly (A) tail, rRNA could not be reversely transcribed in cDNA synthesis using oligo (dT) as a primer [53,54,56].…”

Section: Discussionmentioning

confidence: 99%

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Optimal reference genes for gene expression analysis in polyploid of Cyprinus carpio and Carassius auratus

Liu

Yuan

et al. 2020

BMC Genet

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Background Reference genes are usually stably expressed in various cells and tissues. However, it was reported that the expression of some reference genes may be distinct in different species. In this study, we intend to answer whether the expression of reported traditional reference genes changes or not in the polyploid fish Results By retrieving the mRNA sequencing data of three different ploidy fish from the NCBI SRA database, we selected 12 candidate reference genes, and examined their expression levels in the 10 tissues and in the four cell lines of three different ploidy fish by real-time PCR. Then, the expression profiles of these 12 candidate reference genes were systematically evaluated by using the software platforms: BestKeeper, NormFinder and geNorm. Conclusion The 28S ribosomal protein S5 gene (RPS5) and the ribosomal protein S18 gene (RPS18) are the most suitable reference genes for the polyploid of Cyprinus carpio and Carassius auratus, demonstrated by both of the tissues and the cultured cells.

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“…2). Next, we compared the transcriptional stability of several normalizers that were formerly recommended for use in canine osteosarcoma without transcriptome-wide expression profiling for this context such as OAZ1 [16] or B2M, HNRNPH, RPS5 and RPS19 [17] ( Table S5). This set consisted of classical normalizers that were derived from educated guesswork (e.g.…”

Section: Ranking Mrna-seq Data Based On Stability Of Expressionmentioning

confidence: 99%

S100A4 mRNA-protein relationship uncovered by measurement noise reduction

et al. 2020

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Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and "splicing weakness" involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman's coefficient ρ = 0.72 (p = 0.006)). Key messages• RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour.• Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation.• HNRNPL is stably expressed across various cancer tissues and osteosarcoma.• Logit transformed qIHC score better associates with mRNA amount.• Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.

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Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach

Cited by 22 publications

References 36 publications

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Optimal reference genes for gene expression analysis in polyploid of Cyprinus carpio and Carassius auratus

S100A4 mRNA-protein relationship uncovered by measurement noise reduction

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