Real-Time PCR for mRNA Quantitation

Wong, Marisa L.; Medrano, Juan F.

doi:10.2144/05391rv01

Cited by 1,496 publications

(1,183 citation statements)

References 60 publications

(112 reference statements)

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“…We run real‐time PCR as follows: one cycle at 94°C for 30 s and 40 cycles of denaturation at 94°C for 5 sec, annealing 58°C for 30 sec, and final extension at 72°C for 5 min. We used a BIO‐RAD iQ5 Multicolor Real‐Time Detection System (Bio‐Rad, Hercules, CA, USA) to perform all real‐time PCR reactions and determined the relative expression of the tested reference genes by CT values calculated by 2 −ΔΔCt method (Wong and Medrano 2005). …”

Section: Methodsmentioning

confidence: 99%

Adaptional evolution of trichome in Caragana korshinskii to natural drought stress on the Loess Plateau, China

Ning

Wang

Zhou

et al. 2016

Ecology and Evolution

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Caragana korshinskii is commonly employed to improve drought ecosystems on the Loess Plateau, although the molecular mechanism at work is poorly understood, particularly in terms of the plant's ability to tolerate drought stress. Water is the most severe limiting factor for plant growth on the Loess Plateau. The trichome is known to play an efficient role in reducing water loss through decreasing the rate of transpiration, so in this study, we focused on the trichome‐related gene expression of ecological adaptation in C. korshinskii under low precipitation conditions. In order to explore the responses of trichomes to drought, we selected two experimental sites from wet to dry along the Loess Plateau latitude gradient for observation. Micro‐phenomena through which trichomes grew denser and larger under reduced precipitation were observed using a scanning electron microscope; de novo transcriptomes and quantitative PCR were then used to explore and verify gene expression patterns of C. korshinskii trichomes. Results showed that GIS2,TTG1, and GL2 were upregulated (as key positive‐regulated genes on trichome development), while CPC was downregulated (negative‐regulated gene). Taken together, our data indicate that downstream genes of gibberellin and cytokinin signaling pathways, alongside several cytoskeleton‐related genes, contribute to modulating trichome development to enhance transpiration resistance ability and increase the resistance to drought stress in C. korshinskii.

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Section: Methodsmentioning

confidence: 99%

Adaptional evolution of trichome in Caragana korshinskii to natural drought stress on the Loess Plateau, China

Ning

Wang

Zhou

et al. 2016

Ecology and Evolution

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“…qPCRs were performed using gene-specific primers pairs (Table 1). The DDCt (Cycle threshold) method (Wong and Medrano, 2005) was used to analyse the relative change in gene expression between DM foetuses and their controls and between DM cows and their controls (individual samples were analysed in replicate). The DDCt value for each sample was calculated as follows: DDCt 5 (CtTarget 2 CtReference) in DM animals -(CtTarget 2 CtReference) in control animals.…”

Section: Muscle Samplesmentioning

confidence: 99%

Myostatin inactivation induces a similar muscle molecular signature in double-muscled cattle as in mice

Chelh

Picard

Hocquette

et al. 2011

Animal

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Myostatin (MSTN), a member of the TGF-b superfamily, is a negative regulator of skeletal muscle mass. We have previously shown that the cell survival/apoptosis pathway is a downstream target of MSTN loss-of-function in mice through the regulation of the expression or abundance of many survival and apoptotic factors. In this study, we used western-blot and quantitative PCR (qPCR) analyses to validate these novel downstream targets of MSTN in double-muscled (DM) cattle v. their controls including 260-day-old foetuses and adult cows from the INRA95 strain. MSTN loss-of-function in DM foetuses and DM cows resulted in a glycolytic shift of the muscles (e.g. upregulation of H-MyBP, PGM1 and SNTA1 and downregulation of H-FABP), activation of cell survival pathway through regulation of some components of the PI3K/Akt pathway (e.g. upregulation of DJ-1 and Gsk-3bser9/ Gsk-3btotal ratio and downregulation of PTEN) and upregulation of cell survival factors translationally controlled tumour protein (14-3-3E, Pink1). We also found a lower abundance of pro-apoptotic transcripts and/or proteins (Caspase-3, caspase-8, caspase-9, BID, ID2 and Daxx) and a higher expression of anti-apoptotic transcripts (Traf2 and Bcl2l2) in DM muscles. All together, these results are in favour of activation of the cell survival pathway and loss of apoptosis pathway within the muscles of DM animals. Alteration of both pathways may increase myonuclear or satellite cell survival, which is crucial for protein synthesis. This could contribute to muscle hypertrophy in DM foetuses and DM cows.Keywords: myostatin, cattle, muscle hypertrophy, apoptosis, cell survival ImplicationsMuscle hypertrophy has been extensively studied in meatproducing animals. In double-muscled (DM) cattle, a loss-offunction mutation in the myostatin gene (MSTN ) is responsible for muscle hypertrophy. In order to get a thorough knowledge of the molecular mechanisms of MSTN action, we have examined the molecular phenotype of DM muscle based on our previous data in MSTN-null mice. The data bring new elements to the understanding of the DM muscle phenotype and of the regulation of muscle mass in farm animals.

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“…The relative quantification method was used to evaluate the quantitative real-time PCR data. 50,51 Briefly, this was accomplished by comparing the threshold cycle (C t ) values of gDAF with that of the housekeeping gene to account for differences in quality and quantity between individual samples. Standard curves were generated using serial dilutions from 10 3 to 10 7 concentrations of the external reference sample and were used for amplification efficiency correction.…”

Section: Sequencingmentioning

confidence: 99%

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

et al. 2009

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Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular muscle (EOM) dysfunction among African MG subjects reported earlier may result from altered DAF expression. To test this hypothesis, we screened the DAF gene sequences relevant to the classical complement pathway and found an association between myasthenics with EOM paresis and the DAF regulatory region c.-198C4G SNP (odds ratio ¼ 8.6; P ¼ 0.0003). This single nucleotide polymorphism (SNP) results in a twofold activation of a DAF 5 0 -flanking region luciferase reporter transfected into three different cell lines. Direct matching of the surrounding SNP sequence within the DAF regulatory region with the known transcription factor-binding sites suggests a loss of an Sp1-binding site. This was supported by the observation that the c.-198C4G SNP did not show the normal lipopolysaccharide-induced DAF transcriptional upregulation in lymphoblasts from four patients. Our findings suggest that at critical periods during autoimmune MG, this SNP may result in inadequate DAF upregulation with consequent complement-mediated EOM damage. Susceptible individuals may benefit from anti-complement therapy in addition to immunosuppression.

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Real-Time PCR for mRNA Quantitation

Cited by 1,496 publications

References 60 publications

Adaptional evolution of trichome in Caragana korshinskii to natural drought stress on the Loess Plateau, China

Adaptional evolution of trichome in Caragana korshinskii to natural drought stress on the Loess Plateau, China

Myostatin inactivation induces a similar muscle molecular signature in double-muscled cattle as in mice

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

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