Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR

Sun, Junhong; Nan, Liang-huan; Gao, Cai-rong; Wang, Ying-Yuan

doi:10.1007/s00414-011-0604-3

Cited by 35 publications

(22 citation statements)

References 24 publications

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“…RPL17 is a component of the 60S ribosomal subunit. Although several ribosomal proteins are variably expressed under disease conditions (Kobayashi et al 2006), they have been used as normalization genes (Sun et al 2012). In turbot (S. maximus), RPL17 is the most stable gene across tissue types before bacterial challenge (Dang and Sun 2011).…”

Section: Discussionmentioning

confidence: 99%

Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development

Guo

Gao

et al. 2014

Fish Physiol Biochem

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Real-time reverse transcriptase (RT) polymerase chain reaction (PCR) requires data normalization using an appropriate reference gene in order to obtain more reliable results with biological significance. We cloned a partial sequence of elongation factor-1-α (EF1α) and ribosomal protein L17 (RPL17) from Monopterus albus. We investigated the suitability of five commonly used reference genes [18S ribosomal RNA (18S), cytoskeletal protein (β-actin), glyceraldehyde phosphate dehydrogenase (GAPDH), EF1α and RPL17] as potential quantitative reference genes for normalizing real-time RT-PCR data generated in gonads of different developmental stages and in other tissues of M. albus. Analysis of the data indicated that 18S, β-actin and GAPDH are not suitable as reference genes because of their levels of variations of expression. EF1α and RPL17 might be suitable as reference genes in the gonads of different developmental stages as well as in other tissues of M. albus.

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Section: Discussionmentioning

confidence: 99%

Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development

Guo

Gao

et al. 2014

Fish Physiol Biochem

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“…The stability of transcription of reference genes was assayed with GeNorm system and total six reference genes were analyzed including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β -actin, 18 S rRNA gene, hypoxanthine phosphoribosyltransferase 1-like ( hprt1 ), epinephelus coioides ribosomal protein S29 ( rps29 ) and ribosomal protein L13 ( rpl13 ) [47–49]. GeNorm analysis revealed that rpl13 was the most stable control gene in skeletal muscles (geNorm stability value M = 0.28).…”

Section: Methodsmentioning

confidence: 99%

Daily rhythmicity of clock gene transcript levels in fast and slow muscle fibers from Chinese perch (Siniperca chuatsi)

Cheng

et al. 2016

BMC Genomics

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BackgroundClock genes are considered to be the molecular core of biological clock in vertebrates and they are directly involved in the regulation of daily rhythms in vertebrate tissues such as skeletal muscles. Fish myotomes are composed of anatomically segregated fast and slow muscle fibers that possess different metabolic and contractile properties. To date, there is no report on the characterization of the circadian clock system components of slow muscles in fish.ResultsIn the present study, the molecular clock components (clock, arntl1/2, cry1/2/3, cry-dash, npas2, nr1d1/2, per1/2/3, rorα and tim genes) and their daily transcription levels were characterized in slow and fast muscles of Chinese perch (Siniperca chuatsi). Among the 15 clock genes, nrld2 and per3 had no daily rhythmicity in slow muscles, and cry2/3 and tim displayed no daily rhythmicity in fast muscles of the adult fish. In the slow muscles, the highest expression of the most clock paralogs occurred at the dark period except arntl1, nr1d1, nr1d2 and tim. With the exception of nr1d2 and tim, the other clock genes had an acrophase at the light period in fast muscles. The circadian expression of the myogenic regulatory factors (mrf4 and myf5), mstn and pnca showed either a positive or a negative correlation with the transcription pattern of the clock genes in both types of muscles.ConclusionsIt was the first report to unravel the molecular clock components of the slow and fast muscles in vertebrates. The expressional pattern differences of the clock genes between the two types of muscle fibers suggest that the clock system may play key roles on muscle type-specific tissue maintenance and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3373-z) contains supplementary material, which is available to authorized users.

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“…Different normalization strategies have been employed to reduce variability in the qPCR amplification process including internal reference genes (RGs), normalization to initial amount of material and use of external control nucleotides [3–5]. Applying the use of internal RGs is the most accepted method since expression and amplification of both reference and target genes occur under the same conditions despite different sequences [2].…”

Section: Introductionmentioning

confidence: 99%

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

et al. 2017

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Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

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Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR

Cited by 35 publications

References 24 publications

Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development

Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development

Daily rhythmicity of clock gene transcript levels in fast and slow muscle fibers from Chinese perch (Siniperca chuatsi)

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

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