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  • QRT-PCR: Quantitative Reverse Transcription PCR

    Загружено:

    Madel Tutor Chaturvedi
    30 просмотров19 страниц
    This document discusses quantitative reverse transcription PCR (qRT-PCR). It describes the basic protocol which includes RNA extraction, reverse transcription, amplification, and quantification. Some advantages of qRT-PCR are that it allows analysis of samples with differing target abundance, has little inter-assay variation, and is quantitative. Optimization of qRT-PCR involves choosing the target gene, detection method, oligonucleotides/primers, sample material/processing, and quantification strategy. Primer design considerations and validation of the assay are also discussed.

    Исходное описание:

    presentation on quantitative pcr

    Оригинальное название

    qRT-PCR

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    This document discusses quantitative reverse transcription PCR (qRT-PCR). It describes the basic protocol which includes RNA extraction, reverse transcription, amplification, and quantification. Some advantages of qRT-PCR are that it allows analysis of samples with differing target abundance, has little inter-assay variation, and is quantitative. Optimization of qRT-PCR involves choosing the target gene, detection method, oligonucleotides/primers, sample material/processing, and quantification strategy. Primer design considerations and validation of the assay are also discussed.

    Авторское право:

    © All Rights Reserved
    Скачайте в формате PPTX, PDF, TXT или читайте онлайн в Scribd
    Отметить как неприемлемый контент
    30 просмотров19 страниц

    QRT-PCR: Quantitative Reverse Transcription PCR

    Загружено:

    Madel Tutor Chaturvedi
    This document discusses quantitative reverse transcription PCR (qRT-PCR). It describes the basic protocol which includes RNA extraction, reverse transcription, amplification, and quantification. Some advantages of qRT-PCR are that it allows analysis of samples with differing target abundance, has little inter-assay variation, and is quantitative. Optimization of qRT-PCR involves choosing the target gene, detection method, oligonucleotides/primers, sample material/processing, and quantification strategy. Primer design considerations and validation of the assay are also discussed.

    Авторское право:

    © All Rights Reserved
    Скачайте в формате PPTX, PDF, TXT или читайте онлайн в Scribd
    Отметить как неприемлемый контент
    из 19

    qRT-PCR

    Quantitative Reverse Transcription PCR

    Basic protocol

    RNA
    Extraction

    Reverse
    Transcription

    Amplification

    Quantification

    advantages
    Combination of DNA amplification and
    detection obviates requirements for postPCR processing
    Allows analysis of samples differing in
    target abundance
    Little inter-assay variation
    Quantitative rather than qualitative

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of oligonucleotides and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of oligonucleotides and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of oligonucleotides and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    Detection methods

    amplification
    plot

    Alpha tubulin II
    Std dilution: 5-fold

    Standard curve
    Slope: -3.819
    Efficiency: 98.6

    Melt curve
    Presence of
    multiple species

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of probes and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    Primer design

    Length: 18-24 nt
    Tm: 58-60OC
    GC content: 40-60%
    Amplicon: 100-150 bp
    Avoid secondary structures
    Avoid runs of 4 or more of one base or dinucleotide
    repeats
    Avoid intra- and inter- primer homology

    Primer blast results

    Beacon designer results

    NetPrimer Results

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of oligonucleotides and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    optimization
    Choice of Method
    Choice of target gene
    Choice of detection method
    Choice of oligonucleotides and primers
    Choice of sample material and sample
    processing
    Quantification strategies

    optimization
    Validation
    Verification of design of oligonucleotides
    Verification of amplification
    Optimization of reaction conditions
    PCR characteristics
    Analytical and clinical verification
    Internal quality control
    Proficiency testing

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