A practical approach to RT-qPCR—Publishing data that conform to the MIQE guidelines

Taylor, Sean C.; Wakem, Michael; Dijkman, Greg; Alsarraj, Marwan; Nguyen, Marie

doi:10.1016/j.ymeth.2010.01.005

Cited by 631 publications

(488 citation statements)

References 10 publications

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“…The efficiency of the each primer pairs was evaluated according the equation: E = 10 (-1/slope) for a serial dilution of RNA. The efficiency ranged between 90.49 and 103.40% for the reference genes and target genes, which is within the suggested values (90-110%) for real-time assays [19]). The average expression stability M found by GeNorm point out the GAPDH, S19 as most stable reference genes in our experiment with a M value about 0.36.…”

supporting

confidence: 79%

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Gadiou

Kundu

2012

Indian J. Virol.

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A SYBR Green Ò -based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plantexpressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and bactin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.The fluorescence-based quantitative real-time PCR (qPCR) has been used to detect and measure nucleic acids of a wide range of plant viruses with RNA and DNA genome [13,16,17]. Two main qPCR based techniques: relative and absolute quantification have emerged from various studies on virus detection and quantification of virus titer. The ''absolute quantification'' with known standard is the most commonly used for plant virus quantification. On the other hand, relative quantification uses a comparative quantification with internal reference endogenous housekeeping control genes [1]. The relative quantification determines the changes in RNA levels of a gene across multiple samples and expresses it relative to the levels of an internal control RNA. Therefore, relative quantification does not require standards with known concentrations [15]. Relative quantification employing reference internal control genes was used for several plant virus detection and quantification studies [5,6]. A comparative study showed that the relative quantification assay may be as accurate as absolute quantification when appropriate housekeeping genes are used and normalized with target genes [6,10]. In this paper we have chosen endogenous housekeeping genes for quantification of viruses in apple trees in a relative quantification system. Two apple tree infecting viruses, Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) are selected. ASGV and ApMV belong to the most frequently emerged pathogens encounters in pome fruit trees. Several molecular-based methods have been elaborated to perform routine detection [7,8,11,12]. However, a highly sensitive real-time based detection and quantification assay for these viruses is missing so far. We described here a SYBER green based relative quantification assay employing appropriate endogenous reference genes as internal control.A total of 39 samples coming from different apple cultivars grown in vitro culture (i.e. Idared, Champion, Fragrance) healthy (14 plants) and infected (25 plants) have been used to validate the reference genes as well as the target genes. The leaves of plant material were grinded in liquid nitrogen and the total RNA ...

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confidence: 79%

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Gadiou

Kundu

2012

Indian J. Virol.

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“…A final dissociation step was performed to assess the quality of the amplified product. cDNA from a series of 5-fold dilutions were used for calibration, and the efficiency of the PCR amplifications was found to be in the range of 90% to 110%, which is considered desirable for quantitative PCR (Taylor et al, 2010). Relative expression levels were calculated as the ratio of the target gene transcript level to the transcript level of the housekeeping gene Actin (Smactin; Yang et al, 2010).…”

Section: Gene Expression Analysismentioning

confidence: 99%

Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge

et al. 2015

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“…The Ct value is the cycle number at which the fluorescence signal crosses the designated threshold. These experiments were performed according to the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines (Taylor et al, 2010).…”

Section: Real-time Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Glutamate receptor and transporter modifications in rat organotypic hippocampal slice cultures exposed to oxygen–glucose deprivation: The contribution of cyclooxygenase-2

Llorente

Landucci

Pellegrini‐Giampietro

et al. 2015

Neuroscience

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A practical approach to RT-qPCR—Publishing data that conform to the MIQE guidelines

Cited by 631 publications

References 10 publications

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge

Glutamate receptor and transporter modifications in rat organotypic hippocampal slice cultures exposed to oxygen–glucose deprivation: The contribution of cyclooxygenase-2

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