2000
DOI: 10.1677/jme.0.0250169
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Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Abstract: The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing repr… Show more

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Cited by 3,441 publications
(2,631 citation statements)
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“…Microarray results were validated with real-time quantitative RT-PCR using a strategy described previously [35] with modifications [8,28]. After the last cycle, a dissociation curve was run to check for product purity.…”
Section: Validation Of Microarray Results By Real-time Quantitative Rmentioning
confidence: 99%
“…Microarray results were validated with real-time quantitative RT-PCR using a strategy described previously [35] with modifications [8,28]. After the last cycle, a dissociation curve was run to check for product purity.…”
Section: Validation Of Microarray Results By Real-time Quantitative Rmentioning
confidence: 99%
“…However, although these advantages may be significant, a number of technique and analysis-specific parameters capable of greatly influencing the final result have recently been discussed, 99 including factors such as quality of the template, the reverse-transcription step, selection of endogenous controls, and the data analysis approach employed. Current research has emphasized the need for multiple control genes for relative quantification 100 owing to the inconsistence in expression of what were considered previously as 'housekeeping' genes.…”
Section: Discussionmentioning
confidence: 99%
“…The RiboGreen quantification assay relies on a proprietary dye that exhibits significant fluorescent enhancement upon binding to nucleic acids and that can be detected using a spectrofluorometer, a fluorescence microplate reader or a filter-based fluorometer (Jones et al 1998). At high RNA concentrations, quantification made through absorbance at 260 nm and through the Ribogreen assay are (Bustin 2000). However, at the RNA concentrations typically used for real-time RT-PCR, the RiboGreen assay clearly shows less variability than UV absorbance, allowing a more accurate measurement of lower amounts of RNA (Hashimoto et al 2004).…”
Section: Discussionmentioning
confidence: 99%
“…The ideal internal standard should be expressed at a constant level among different tissues of an organism, at all stages of development, and should be unaffected by the experimental treatment (Bustin 2000). However, it is unlikely that such a gene would exist since biological systems are dynamic and constantly changing in response to their environment.…”
Section: Introductionmentioning
confidence: 99%