Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro

Winer, Jane; Jung, Chang Jin; Shackel, I.; Williams, P. Mickey

doi:10.1006/abio.1999.4085

Cited by 1,287 publications

(927 citation statements)

References 14 publications

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“…24 However, unless the variability is defined this argument simply does not hold. While genes like GAPDH have been found to be appropriate for certain experimental situations 25 21 who demonstrated that if the wrong reference gene is chosen it can result in altered findings. This occurs when the reference gene is regulated by the experimental conditions.…”

Section: Normalisation; Reference Genesmentioning

confidence: 99%

Real-time RT-PCR normalisation; strategies and considerations

et al. 2005

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Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.

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Section: Normalisation; Reference Genesmentioning

confidence: 99%

Real-time RT-PCR normalisation; strategies and considerations

et al. 2005

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“…The carp β-actin and zebrafish β-actin were used as the endogenous control. All samples were normalized to internal controls and fold changes were calculated by relative quantification (2 −ΔΔCt ) (Livak and Schmittgen, 2001;Winer et al, 1999;Xiao et al, 2014). For the analysis of proTα b and Tβ-l expression levels, the gill was used as calibrator organ for analyzing differential expression in organs from healthy fish, the organs from the control fish were used as calibrators for analyzing of the expression in virus-infected fish, the 4 h pf carp embryos were used as the calibrator for analyzing the expression in the early development.…”

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

Characterization of two thymosins as immune-related genes in common carp (Cyprinus carpio L.)

Xiao

Shen

Hong

et al. 2015

Developmental & Comparative Immunology

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“…All samples were examined in duplicate and 18S ribosomal RNA (Applied Biosystems, Foster City, CA) was used as an endogenous control. The data were analyzed using the CT method that has been shown to produce comparable results to an absolute quantification procedure [27] and has been previously used to examine cytokine mRNA levels in skeletal muscle [28,29]. Two different fluorescent dyes were utilized to examine the genes of interest (FAM) and the 18S RNA control (VIC).…”

Section: Quantitative Real-time Pcr Verification Of Rt-pcrmentioning

confidence: 99%

Death receptor-associated pro-apoptotic signaling in aged skeletal muscle

2006

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Tumor necrosis factor-alpha (TNF-α) is elevated in the serum as a result of aging and it promotes pro-apoptotic signaling upon binding to the type I TNF receptor. It is not known if activation of this apoptotic pathway contributes to the well-documented age-associated decline in muscle mass (i.e. sarcopenia). We tested the hypothesis that skeletal muscles from aged rodents would exhibit elevations in markers involved in the extrinsic apoptotic pathway when compared to muscles from young adult rodents, thereby contributing to an increased incidence of nuclear apoptosis in these muscles. The plantaris (fast) and soleus (slow) muscles were studied in young adult (5-7 mo, n = 8) and aged (33 mo, n = 8) Fischer 344 × Brown Norway rats. Muscles from aged rats were significantly smaller while exhibiting a greater incidence of apoptosis. Furthermore, muscles from aged rats had higher type I TNF receptor and Fas associated death domain protein (FADD) mRNA, protein contents for FADD, BCL-2 Interacting Domain (Bid), FLICE-inhibitory protein (FLIP), and enzymatic activities of caspase-8 and caspase-3 than muscles from young adult rats. Significant correlations were observed in the plantaris muscle between caspase activity and muscle weight and the apoptotic index, while similar relationships were not found in the soleus. These data demonstrate that pro-apoptotic signaling downstream of the TNF receptor is active in aged muscles. Furthermore, our data extend the previous demonstration that type II fibers are preferentially affected by aging and support the hypothesis that type II fiber containing skeletal muscles may be more susceptible to muscle mass loses via the extrinsic apoptotic pathway.

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Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro

Cited by 1,287 publications

References 14 publications

Real-time RT-PCR normalisation; strategies and considerations

Real-time RT-PCR normalisation; strategies and considerations

Characterization of two thymosins as immune-related genes in common carp (Cyprinus carpio L.)

Death receptor-associated pro-apoptotic signaling in aged skeletal muscle

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