Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies

Peters, Iain R.; Peeters, Dominique; Helps, Christopher R; Day, Michael

doi:10.1016/j.vetimm.2007.01.011

Cited by 186 publications

(141 citation statements)

References 26 publications

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“…Over the past few decades, it was demonstrated that the use of a single reference appears to be insufficient in the normalization of qPCR data (Tricarico et al 2002;Vandesompele et al 2002;Pfaffl et al 2004), and the use of multiple references was recommended for qPCR data normalization (Brinkhof et al 2006;Peters et al 2007;Varshney et al 2012). Using geNorm algorithm, therefore, we further determined the optimal number of reference RNAs required for data normalization of miRNA quantitation through calculating the pairwise variation (V n/n+1 ) between each combination of sequential normalization factors (NF n and NF n+1 ).…”

Section: Resultsmentioning

confidence: 99%

Combination of let-7d-5p, miR-26a-5p, and miR-15a-5p is suitable normalizer for studying microRNA expression in skin tissue of Liaoning cashmere goat during hair follicle cycle

Bai¹,

Dang²,

Yin³

et al. 2016

CZECH J ANIM SCI

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ABSTRACT:The microRNAs are non-coding RNA molecules of approximately 20-22 nucleotides that are found to be implicated in a wide range of physiological processes. In this study, the suitability of 10 candidate reference RNAs was evaluated for microRNA expression data in the skin tissue of Liaoning cashmere goat including 1 small nuclear RNA (snRNA; RNU6B), 1 small nucleolar RNA (snoRNA; Z30), 1 rRNA (5S), 1 transfer RNA (tRNA; MettRNA), and 6 microRNAs (miR; let-7d-5p, miR-15a-5p, miR-26a-5p, miR-125a-5p, miR-214-3p, and miR-221-3p). Based on geNorm and NormFinder algorithms, we identified let-7d-5p, miR-26a-5p, and miR-15a-5p as the most stable reference RNAs. Also, three reference RNAs (let-7d-5p, miR-26a-5p, and miR-15a-5p) were sufficient for the normalization of microRNA expression data in the skin of this breed. We further assessed the suitability of let-7d-5p, miR-26a-5p, and miR-15a-5p in a combination as reference RNAs through detecting the relative expression of miR- 24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p as putative genes of interest. Significant differences were revealed in the relative expression of miR- 24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p at telogen stage of hair follicle cycle when a combination of let-7d-5p, miR-26a-5p, and miR-15a-5p vs a single let-7d-5p were used as reference RNA. Based on the results from this study, we suggested that the combination of let-7d-5p, miR-26a-5p, and miR-15a-5p as normalizers for microRNA expression data would be more reliable than that of single let-7d-5p, and the geometric mean of these three microRNAs (let-7d-5p, miR-26a-5p, and miR15a-5p) can be used for the normalization of microRNAs expression data in the skin of Liaoning cashmere goat.

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Section: Resultsmentioning

confidence: 99%

Combination of let-7d-5p, miR-26a-5p, and miR-15a-5p is suitable normalizer for studying microRNA expression in skin tissue of Liaoning cashmere goat during hair follicle cycle

Bai¹,

Dang²,

Yin³

et al. 2016

CZECH J ANIM SCI

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“…RPL32 gene was selected as an ideal reference gene in the majority of canine tissues including muscle. RPS18 was the gene with the most stable expression in canine lungs and spleen, and it is also a suitable reference gene in muscle tissue (Peters et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

Nesvadbová¹,

Knoll²

2011

CZECH J ANIM SCI

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ABSTRACT:The selection of reference genes is essential for gene expression studies when using a realtime quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.

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“…Recent studies showed that most of the reference genes could also change the expression levels under internal or external factors, so there was no universal internal control gene that can be applied for normalization of gene expression under all conditions (Peters et al, 2007;Mitter et al, 2009). What we can do and should do was screening relative most stable reference genes to keep the results of gene expression with higher reliability.…”

Section: Introductionmentioning

confidence: 99%

Selection of Reliable Reference Genes for Quantitative Real-Time PCR in Golden-Line Barbell (Sinocyclocheilus grahami) During Juvenile and Adult Stages

yuanwei¹,

Pan²,

Wang³

et al. 2017

FisheriesSciences.com

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Abstract:In order to obtain reliable results of gene expression, appropriate internal control gene(s) were required for normalization before real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Here, we selected eight candidate housekeeping genes as the potential reference genes in normalizing qRT-PCR data in ten tissues of the juvenile and adult stages of a naturally tetraploid cyprinid fish Sinocyclocheilus grahami. Before qRT-PCR, appropriate primers were designed and compared for distinguishing different copies in some duplicate genes. Candidate reference genes were evaluated for their stability using online software RefFinder, which is integrated by four normal software of the comparative delta-C T , BestKeeper, NormFinder and GeNorm. According to the rankings produced by these analyses, Eef2, ACTB and G6PD were the most stable reference genes as internal controls for qRT-PCR in juvenile, while B2M, Eef2 and RPL17 were shown to be most appropriate in adult fish. All the analyses show that GAPDH was the least stable expression gene which would be unsuitable as reference gene at both stages. This is the first report about reference gene selection in Sinocyclocheilus and we think that is beneficial for the future gene expression studies in golden-line barbel.

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Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies

Cited by 186 publications

References 26 publications

Combination of let-7d-5p, miR-26a-5p, and miR-15a-5p is suitable normalizer for studying microRNA expression in skin tissue of Liaoning cashmere goat during hair follicle cycle

Combination of let-7d-5p, miR-26a-5p, and miR-15a-5p is suitable normalizer for studying microRNA expression in skin tissue of Liaoning cashmere goat during hair follicle cycle

Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

Selection of Reliable Reference Genes for Quantitative Real-Time PCR in Golden-Line Barbell (Sinocyclocheilus grahami) During Juvenile and Adult Stages

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