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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...
67.6K

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A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae
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Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

April M Lewis1, Kelly C Rice2

  • 1Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, 32611-0700, USA.

Methods in Molecular Biology (Clifton, N.J.)
|February 4, 2015
PubMed
Summary

Quantitative real-time PCR (qPCR) quantifies RNA transcripts to compare gene expression in biological samples. This method details a two-step qPCR workflow for calculating relative gene expression changes in S. aureus.

Keywords:
Livak calculationRNAReal-time PCRRelative quantificationSYBR GreencDNA

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Quantitative real-time PCR (qPCR) is essential for measuring gene expression levels.
  • Accurate quantification of RNA transcripts is crucial for understanding biological processes and disease mechanisms.
  • Standardized methods are needed for reliable gene expression analysis in bacterial pathogens like S. aureus.

Purpose of the Study:

  • To describe a two-step quantitative real-time PCR (qPCR) method for assessing gene expression.
  • To enable the calculation of relative fold-change in gene expression for genes of interest in S. aureus.
  • To provide a workflow for reliable gene expression analysis in bacterial research.

Main Methods:

  • RNA extraction and quality control to ensure the absence of genomic DNA contamination.
  • Two-step cDNA synthesis from RNA templates using random primers and reverse-transcriptase.
  • SYBR Green-based quantitative real-time PCR (qPCR) for gene expression analysis.

Main Results:

  • The described workflow allows for the synthesis of cDNA from RNA templates.
  • SYBR Green-based qPCR enables the quantification of specific gene expression.
  • Relative fold-change in gene expression can be accurately calculated for target genes in S. aureus.

Conclusions:

  • The two-step qPCR method provides a sensitive approach for quantifying gene expression in S. aureus.
  • This workflow facilitates the comparison of gene expression levels across different biological samples.
  • The method is valuable for researchers studying gene regulation and function in bacterial pathogens.