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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

Manuel Ares

    Cold Spring Harbor Protocols
    |October 3, 2014
    PubMed
    Summary
    This summary is machine-generated.

    Quantitative polymerase chain reaction (qPCR) measures DNA molecules using fluorescence. This method quantifies specific DNA sequences in samples, enabling relative or absolute quantification for research applications.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Quantitative polymerase chain reaction (qPCR) is a widely used molecular biology technique.
    • It allows for the detection and quantification of specific DNA sequences.
    • Understanding the principles of qPCR is crucial for accurate molecular analysis.

    Purpose of the Study:

    • To describe a protocol for measuring DNA molecules using quantitative polymerase chain reaction (qPCR).
    • To explain the principles of fluorescence-based PCR amplification and real-time detection.
    • To outline methods for relative and absolute quantification of target DNA sequences.

    Main Methods:

    • Utilizes fluorescence-based PCR amplification to detect DNA.
    • Monitors the accumulation of dsDNA-dye complexes in real time.
    • Employs threshold cycle (Ct) determination for quantification.

    Main Results:

    • qPCR allows for the exponential amplification of target DNA sequences.
    • Real-time fluorescence measurement enables monitoring of reaction progress.
    • The threshold cycle (Ct) value correlates with the initial amount of target DNA.

    Conclusions:

    • qPCR provides a sensitive and accurate method for quantifying DNA molecules.
    • The protocol supports both relative quantification against a reference sequence and absolute quantification using a standard curve.
    • This technique is valuable for various applications in molecular biology and diagnostics.