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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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Related Experiment Video

Updated: Apr 14, 2026

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

Absolute Quantification of microRNA Copies in Exosomes Using Real-Time PCR.

Pragati Raghuwanshi¹, Prasenjit Srivastava¹, Nishant Singh¹

¹All India Institute of Medical Sciences (AIIMS), Raebareli, India.

Current Protocols

|April 13, 2026

Summary

This summary is machine-generated.

This study presents a novel assay to quantify microRNA (miRNA) molecules within exosomes. The method combines nanoparticle tracking analysis (NTA) and qPCR for precise exosome and miRNA measurement, advancing diagnostic and therapeutic applications.

Keywords:

exosomes miRNAs nanoparticle tracking analysis qPCR standard curve

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Last Updated: Apr 14, 2026

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

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Published on: April 14, 2015

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

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Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Published on: June 14, 2013

Area of Science:

Extracellular Vesicle Biology
Molecular Diagnostics
Biomarker Discovery

Background:

Exosomes, small extracellular vesicles abundant in serum, mediate intercellular communication via encapsulated biomolecules.
Accurate quantification of specific microRNAs (miRNAs) within exosomes is crucial for understanding their biological roles and for developing diagnostic and therapeutic tools.
Current methods lack the precision needed for absolute miRNA quantification per exosome.

Purpose of the Study:

To develop and validate a robust assay for the absolute quantification of miRNA copy number per exosome.
To establish a standardized workflow for exosome isolation, characterization, and miRNA analysis from human serum.
To provide a framework for advancing exosome-based biomarker and therapeutic development.

Main Methods:

Exosome isolation from human serum via ultracentrifugation.
Nanoparticle Tracking Analysis (NTA) for exosome concentration and size determination.
Absolute quantification of miRNA using standard curve-based quantitative PCR (qPCR) after optimized RNA extraction and cDNA synthesis.

Main Results:

A comprehensive workflow enabling the calculation of miRNA copy number per exosome.
Demonstrated reproducibility and accuracy across diverse biological samples.
Established protocols for vesicle isolation, particle measurement, RNA extraction, and absolute miRNA quantification.

Conclusions:

The developed exosome copy number assay provides precise molecular quantification, essential for exosome biology research.
This method supports the development of exosome-based diagnostics and therapeutics by enabling accurate biomarker assessment.
The protocol offers a practical framework for reproducible exosome analysis and advancing the field of extracellular vesicle research.