Real Time PCR and qPCR Techniques

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.