30 Questions
What does Cycle Threshold (Ct) indicate in qPCR?
The cycle number at which fluorescence reaches threshold line
What is the purpose of Absolute quantitation in qPCR?
Requires a standard of known concentration for Reference Gene (RG) and generating a standard curve for quantitation
What does Relative quantitation in qPCR calculate?
The ratio of Reference Gene (RG) and Target Gene
What is the primary use of qPCR in pharmaceutical biotechnology?
Quality control in manufacturing, viral safety testing, pharmacokinetics and pharmacodynamics studies, pharmaceutical research, and toxicology studies
What does RT-PCR allow in terms of template usage?
RNA to be used as template through cDNA synthesis
What is the crucial step before reverse transcription in qPCR?
RNA isolation using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample
What does reverse transcription do in qPCR?
Synthesizes cDNA from RNA
What is the primary application of qPCR in gene expression analysis?
Gene expression analysis in cancer patients
What does Negative control represent in qPCR?
Successful absence of fluorescence below threshold line
What type of column is used for RNA isolation before reverse transcription?
Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample
What is Real Time PCR?
A modification of conventional PCR where product accumulation is quantified in real time
What is the basis of quantitation in qPCR?
A curve with initiation, exponential, and plateau phases
What is the function of a TaqMan® probe in qPCR?
To bind downstream of the qPCR primers and emit a fluorescent signal upon cleavage
What does the SYBR® Green fluorescent dye do in qPCR?
Binds nonspecifically to dsDNA as it is generated and emits fluorescence upon intercalating with newly synthesized DNA
What is the role of the optical module in qPCR?
For detection of fluorescence emission during the reaction
What does Real Time or Quantitative (qPCR) determine?
The amount of target sequence or gene that is present in a sample
What is the purpose of the TaqMan® probe in qPCR?
To emit a fluorescent signal as DNA polymerase extends the primer
What is the basis of quantitation in qPCR?
A curve with initiation, exponential, and plateau phases
What does the SYBR® Green dye do in qPCR?
Binds nonspecifically to dsDNA and emits fluorescence as DNA is generated
What is Real Time PCR?
A modification of conventional PCR where product accumulation is quantified 'in real time'
What does Cycle Threshold (Ct) indicate in qPCR?
The cycle number at which the fluorescent signal exceeds the background level
What does Negative control represent in qPCR?
A sample containing no template DNA to check for contamination or non-specific amplification
What is the function of a TaqMan® probe in qPCR?
To emit a fluorescent signal as DNA polymerase extends the primer
What does RT-PCR allow in terms of template usage?
It allows for amplification and quantitation of RNA template
What is the purpose of Absolute quantitation in qPCR?
To determine the exact quantity of the target sequence using a standard curve
What does Cycle Threshold (Ct) indicate in qPCR?
The cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence
What is the crucial step before reverse transcription in qPCR?
RNA isolation using Oligo d(T) Affinity chromatomy column to enrich mRNAs and quantify RNA sample
What does Real Time or Quantitative (qPCR) determine?
The cycle number at which fluorescence reaches threshold line, lower values indicate high abundance of target sequence
What is the function of a TaqMan® probe in qPCR?
To cleave and release a fluorescent reporter dye, resulting in increased fluorescence signal
What type of column is used for RNA isolation before reverse transcription?
Oligo d(T) Affinity chromatomy column to enrich mRNAs
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.
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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