Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

By Stephen A. Bustin. Journal of Molecular Endocrinology, 2002

2,615 citations recorded by the source corpus.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/5584f6103597d50d6222cb342c3dd202b098385b

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In Molecular Biology

1. Sheng Zhao
2. Russell D. Fernald
3. T. Nolan
4. W. Scheible
5. M. W. Pfaffl
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7. R. Hands
8. Mark Stitt

Citations in the corpus

• Generation of expressed sequence tags (ESTs) for gene discovery and marker development in cultivated peanut (2005, methodology): “RT-PCR analysis standing the complexity of gene expression and regu-was conducted to validate the microarray and EST data (Bustin, 2002) and to detect the respective expression lation.”
• Validating internal controls for quantitative plant gene expression studies (2004, methodology): “In addition, there can be imbalances in rRNA and mRNA fractions between different samples, and 18S is not always expressed at a constant level in all conditions [1].”
• Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces (2005, background): “It requires only a very small amount of RNA sample (as low as 200 pg), the use of a size standard during electrophoresis allows the estimation of sizes of RNA bands and the measurement appears relatively unaffected by contaminants.”
• Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. (2006, background): “In comparison to classical reverse transcription-polymerase chain reaction (RT-PCR), the main advantages of real-time PCR are its higher sensitivity, specificity, and broad quantification range of up to seven orders of magnitude [1,2].”
• Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. (2004, background): “23 Assays using DNA-binding dyes have two advantages over probe-based ones: (1) they can be incorporated into optimized and long-established protocols that use legacy primers and experimental conditions, and (2) they are significantly cheaper, as there is no probe-associated cost.”
• Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi (2004, methodology): “The new quenchers are defined as dark quenchers or black hole quenchers (BHQ) and are suitable for a wide range of dyes since they can quench fluorescence over the entire visible spectrum and into the infrared (Bustin, 2002).”
• The power of real-time PCR. (2005, background): “Of course, conclusions based on data derived from real-time PCR are best utilized when the biological context is well understood ( 10 ).”
• Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula (2006, neutral): “be included [32].”
• A standard curve based method for relative real time PCR data processing (2005, methodology): “Summarizing data from several reference genes Several reference genes are required for accurate relative quantification [1,20].”
• Quantification of mRNA using real-time RT-PCR (2006, result): “and (iii) data analysis is highly subjective and, if carried out inappropriately, obfuscates the actual results obtained from the assa”