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Real Time RT-PCR

Real Time RT-PCR

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...

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Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Published on: January 22, 2014

Cerebrospinal fluid MicroRNA profiling using quantitative real time PCR.

Marco Pacifici¹, Serena Delbue, Ferdous Kadri

¹Medical School and Stanley S. Scott Cancer Center, LSU Health Sciences Center.

Journal of Visualized Experiments : Jove

|February 12, 2014

Summary

This study presents a sensitive quantitative real-time PCR method for profiling microRNAs (miRNAs) in cerebrospinal fluid. This technique enables the detection of miRNA expression profiles for potential biomarker discovery in neurological diseases.

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

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Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

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Published on: January 22, 2014

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Published on: August 3, 2011

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Published on: April 14, 2015

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

MicroRNAs (miRNAs) are key regulators of gene expression, influencing translational repression of messenger RNAs (mRNAs).
Altered miRNA expression is implicated in complex diseases, and circulating miRNAs in body fluids show stability and potential as biomarkers.
Cerebrospinal fluid (CSF) miRNA profiling offers a promising avenue for identifying disease-specific signatures.

Purpose of the Study:

To describe a sensitive quantitative real-time PCR (qPCR) method for profiling microRNAs (miRNAs) in cerebrospinal fluid (CSF).
To establish a reliable protocol for miRNA detection in various biological samples, including CSF, for biomarker analysis.

Main Methods:

Utilized Exiqon microRNA ready-to-use PCR human panels (I and II V2.R) for detecting 742 unique human miRNAs.
Performed quantitative real-time PCR (qPCR) assays in triplicate runs.
Processed and analyzed data using GenEx Professional 5 software.

Main Results:

Successfully established and validated a sensitive qPCR protocol for miRNA profiling.
Demonstrated the protocol's efficacy across diverse sample types: cell lines, primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.
The method allows for the detection of a broad range of human miRNAs.

Conclusions:

Quantitative real-time PCR provides a sensitive and effective method for profiling microRNAs in cerebrospinal fluid.
This validated protocol supports the identification of miRNA expression profiles in CSF, crucial for biomarker discovery in neurodegenerative disorders and other conditions.
The protocol's versatility extends to various sample types, enhancing its utility in biomedical research.