Real Time PCR and qPCR Techniques
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Questions and Answers

What does Cycle Threshold (Ct) indicate in qPCR?

  • The cycle number at which PCR amplification begins
  • The cycle number at which PCR amplification ends
  • The cycle number at which fluorescence reaches threshold line (correct)
  • The cycle number at which fluorescence is detected

    • What is the purpose of Absolute quantitation in qPCR?

  • Allows RNA to be used as template through cDNA synthesis
  • Calculates the ratio of RG and Target Gene
  • Requires a standard of known concentration for Reference Gene (RG) and generating a standard curve for quantitation (correct)
  • Used for gene expression analysis in cancer patients

    • What does Relative quantitation in qPCR calculate?

  • The cycle number at which PCR amplification begins
  • The cycle number at which fluorescence reaches threshold line
  • The cycle number at which PCR amplification ends
  • The ratio of Reference Gene (RG) and Target Gene (correct)

    • What is the primary use of qPCR in pharmaceutical biotechnology?

    <p>Quality control in manufacturing, viral safety testing, pharmacokinetics and pharmacodynamics studies, pharmaceutical research, and toxicology studies</p> Signup and view all the answers

    What does RT-PCR allow in terms of template usage?

    <p>RNA to be used as template through cDNA synthesis</p> Signup and view all the answers

    What is the crucial step before reverse transcription in qPCR?

    <p>RNA isolation using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample</p> Signup and view all the answers

    What does reverse transcription do in qPCR?

    <p>Synthesizes cDNA from RNA</p> Signup and view all the answers

    What is the primary application of qPCR in gene expression analysis?

    <p>Gene expression analysis in cancer patients</p> Signup and view all the answers

    What does Negative control represent in qPCR?

    <p>Successful absence of fluorescence below threshold line</p> Signup and view all the answers

    What type of column is used for RNA isolation before reverse transcription?

    <p>Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample</p> Signup and view all the answers

    What is Real Time PCR?

    <p>A modification of conventional PCR where product accumulation is quantified in real time</p> Signup and view all the answers

    What is the basis of quantitation in qPCR?

    <p>A curve with initiation, exponential, and plateau phases</p> Signup and view all the answers

    What is the function of a TaqMan® probe in qPCR?

    <p>To bind downstream of the qPCR primers and emit a fluorescent signal upon cleavage</p> Signup and view all the answers

    What does the SYBR® Green fluorescent dye do in qPCR?

    <p>Binds nonspecifically to dsDNA as it is generated and emits fluorescence upon intercalating with newly synthesized DNA</p> Signup and view all the answers

    What is the role of the optical module in qPCR?

    <p>For detection of fluorescence emission during the reaction</p> Signup and view all the answers

    What does Real Time or Quantitative (qPCR) determine?

    <p>The amount of target sequence or gene that is present in a sample</p> Signup and view all the answers

    What is the purpose of the TaqMan® probe in qPCR?

    <p>To emit a fluorescent signal as DNA polymerase extends the primer</p> Signup and view all the answers

    What is the basis of quantitation in qPCR?

    <p>A curve with initiation, exponential, and plateau phases</p> Signup and view all the answers

    What does the SYBR® Green dye do in qPCR?

    <p>Binds nonspecifically to dsDNA and emits fluorescence as DNA is generated</p> Signup and view all the answers

    What is Real Time PCR?

    <p>A modification of conventional PCR where product accumulation is quantified 'in real time'</p> Signup and view all the answers

    What does Cycle Threshold (Ct) indicate in qPCR?

    <p>The cycle number at which the fluorescent signal exceeds the background level</p> Signup and view all the answers

    What does Negative control represent in qPCR?

    <p>A sample containing no template DNA to check for contamination or non-specific amplification</p> Signup and view all the answers

    What is the function of a TaqMan® probe in qPCR?

    <p>To emit a fluorescent signal as DNA polymerase extends the primer</p> Signup and view all the answers

    What does RT-PCR allow in terms of template usage?

    <p>It allows for amplification and quantitation of RNA template</p> Signup and view all the answers

    What is the purpose of Absolute quantitation in qPCR?

    <p>To determine the exact quantity of the target sequence using a standard curve</p> Signup and view all the answers

    What does Cycle Threshold (Ct) indicate in qPCR?

    <p>The cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence</p> Signup and view all the answers

    What is the crucial step before reverse transcription in qPCR?

    <p>RNA isolation using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample</p> Signup and view all the answers

    What does Real Time or Quantitative (qPCR) determine?

    <p>The cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence</p> Signup and view all the answers

    What is the function of a TaqMan® probe in qPCR?

    <p>To cleave and release a fluorescent reporter dye, resulting in increased fluorescence signal</p> Signup and view all the answers

    What type of column is used for RNA isolation before reverse transcription?

    <p>Oligo d(T) Affinity chromatomy column to enrich mRNAs</p> Signup and view all the answers

    Study Notes

    • PCR instrument can automatically or manually set the threshold for detecting fluorescence signals

    • Negative control represents successful absence of fluorescence below threshold line

    • Cycle Threshold (Ct) is the cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence

    • Absolute quantitation requires a standard of known concentration for Reference Gene (RG) and generating a standard curve for quantitation

    • Relative quantitation calculates the ratio of RG and Target Gene, accuracy depends on RG stability and method used is delta delta Ct (2-ΔΔCq)

    • In pharmaceutical biotechnology, qPCR is used for quality control in manufacturing, viral safety testing, pharmacokinetics and pharmacodynamics studies, pharmaceutical research, and toxicology studies

    • RT-PCR allows RNA to be used as template through cDNA synthesis, taking advantage of poly A tail in mRNAs

    • RNA isolation is crucial, using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample

    • Reverse transcription synthesizes cDNA from RNA, and PCR amplifies it, cDNA quality is assessed using various techniques such as gel electrophoresis and spectrophotometry.

    • qPCR is used for gene expression analysis in cancer patients, identifying viral load in samples, and amplifying specific DNA regions in forensic investigations.

    • PCR instrument can automatically or manually set the threshold for detecting fluorescence signals

    • Negative control represents successful absence of fluorescence below threshold line

    • Cycle Threshold (Ct) is the cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence

    • Absolute quantitation requires a standard of known concentration for Reference Gene (RG) and generating a standard curve for quantitation

    • Relative quantitation calculates the ratio of RG and Target Gene, accuracy depends on RG stability and method used is delta delta Ct (2-ΔΔCq)

    • In pharmaceutical biotechnology, qPCR is used for quality control in manufacturing, viral safety testing, pharmacokinetics and pharmacodynamics studies, pharmaceutical research, and toxicology studies

    • RT-PCR allows RNA to be used as template through cDNA synthesis, taking advantage of poly A tail in mRNAs

    • RNA isolation is crucial, using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample

    • Reverse transcription synthesizes cDNA from RNA, and PCR amplifies it, cDNA quality is assessed using various techniques such as gel electrophoresis and spectrophotometry.

    • qPCR is used for gene expression analysis in cancer patients, identifying viral load in samples, and amplifying specific DNA regions in forensic investigations.

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    Related Documents

    qPCR and RT-PCR Lecture Notes PDF

    Description

    Test your knowledge on Real Time PCR (qPCR) techniques, a modification of conventional PCR used for quantifying DNA product accumulation in real time as the reaction progresses.

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