Source 1review2000Journal of Molecular Endocrinology
Toolkit/reverse transcription polymerase chain reaction
reverse transcription polymerase chain reaction
Also known as: conventional RT-PCR, RT-PCR
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
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.
Usefulness & Problems
Why this is useful
RT-PCR is described as a highly sensitive method for detecting low-abundance mRNA. In this review it serves as the conventional baseline against which kinetic real-time methods are compared.; detection of low-abundance mRNA; analysis of limited tissue samples
Source:
RT-PCR is described as a highly sensitive method for detecting low-abundance mRNA. In this review it serves as the conventional baseline against which kinetic real-time methods are compared.
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detection of low-abundance mRNA
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analysis of limited tissue samples
Problem solved
It enables detection of low-abundance mRNA from limited tissue samples.; sensitive detection of scarce mRNA transcripts
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It enables detection of low-abundance mRNA from limited tissue samples.
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sensitive detection of scarce mRNA transcripts
Problem links
sensitive detection of scarce mRNA transcripts
LiteratureIt enables detection of low-abundance mRNA from limited tissue samples.
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It enables detection of low-abundance mRNA from limited tissue samples.
Published Workflows
Objective: Obtain accurate and reproducible quantitative measurements of mRNA transcription using real-time RT-PCR assays.
Why it works: The review states that fluorescence-based kinetic RT-PCR simplifies reproducible quantification, but only when practical problems are understood and the assay is carefully designed, applied, and validated.
fluorescence-based kinetic monitoring of PCR amplificationabsolute quantification of mRNAreal-time RT-PCRcomparison of conventional versus kinetic RT-PCR systemsexperimental designassay validation
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Techniques
Functional AssayTarget processes
transcriptionImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
The method requires reverse transcription and PCR amplification of RNA-derived templates. The review indicates that quantitative use also requires careful design and validation.; quantitative use is limited by PCR-associated issues; requires careful design and validation when used for quantitation
The review states that conventional RT-PCR has substantial problems in true sensitivity, reproducibility, specificity, and quantitative reliability.; complex technique; substantial problems with true sensitivity, reproducibility and specificity; as a quantitative method, suffers from problems inherent in PCR
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Fluorescence-based kinetic real-time RT-PCR procedures significantly simplify reproducible mRNA quantification and are presented as promising ways to overcome limitations of conventional RT-PCR.
Conventional RT-PCR has substantial problems in true sensitivity, reproducibility, specificity, and quantitative reliability because it inherits problems inherent in PCR.
The review illustrates that transcription levels of the housekeeping gene GAPDH can differ significantly between individuals, indicating caution in assuming uniform housekeeping-gene expression.
RT-PCR is described as a highly sensitive method for detecting low-abundance mRNA from limited tissue samples.
Accurate quantitative transcription measurements by real-time RT-PCR require clear understanding of practical problems plus careful experimental design, application, and validation.
Approval Evidence
1 source3 linked approval claimsfirst-pass slug reverse-transcription-polymerase-chain-reaction
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.
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comparative method summarysupports
Fluorescence-based kinetic real-time RT-PCR procedures significantly simplify reproducible mRNA quantification and are presented as promising ways to overcome limitations of conventional RT-PCR.
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limitation summarysupports
Conventional RT-PCR has substantial problems in true sensitivity, reproducibility, specificity, and quantitative reliability because it inherits problems inherent in PCR.
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performance summarysupports
RT-PCR is described as a highly sensitive method for detecting low-abundance mRNA from limited tissue samples.
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Comparisons
Source-stated alternatives
The main alternative discussed in the abstract is fluorescence-based kinetic real-time RT-PCR.
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The main alternative discussed in the abstract is fluorescence-based kinetic real-time RT-PCR.
Source-backed strengths
described as the most sensitive method for detection of low-abundance mRNA
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described as the most sensitive method for detection of low-abundance mRNA
The main alternative discussed in the abstract is fluorescence-based kinetic real-time RT-PCR.
Shared frame: source-stated alternative in extracted literature
Strengths here: described as the most sensitive method for detection of low-abundance mRNA.
Relative tradeoffs: complex technique; substantial problems with true sensitivity, reproducibility and specificity; as a quantitative method, suffers from problems inherent in PCR.
Source:
The main alternative discussed in the abstract is fluorescence-based kinetic real-time RT-PCR.
Ranked Citations
- 1.