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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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Updated: Jun 16, 2026

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Published on: June 26, 2016

Digital quantification of gene expression using emulsion PCR.

Xiaolong Shi¹, Chao Tang, Wei Wang

¹State Key Laboratory of Bioelectronics, Southeast University, Nanjing, PR China.

Electrophoresis

|February 2, 2010

Summary

This summary is machine-generated.

This study introduces a novel mRNA quantification assay using mRNA mediate-ligation and BEAMing. The technique accurately measures multiple gene transcripts in small cell samples, revealing Sox5 downregulation after specific treatment.

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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Published on: February 25, 2017

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Accurate quantification of mRNA levels is crucial for understanding gene expression.
Existing methods may require larger cell quantities or lack parallel measurement capabilities.
Single-molecule techniques offer high sensitivity for gene expression analysis.

Purpose of the Study:

To develop and validate a single-molecule assay for accurate, parallel mRNA quantification.
To assess the utility of the assay in a biological context, such as gene expression changes in leukemia cells.
To enable precise measurement of multiple mRNA targets from limited cellular material.

Main Methods:

Utilized mRNA mediate-ligation coupled with BEAMing (beads, emulsion, amplification, and magnetics).
Employed pairs of oligos to target specific mRNA transcripts for ligation.
Clonally amplified ligated products on beads within emulsion droplets for detection.
Quantified mRNA levels by counting amplified beads immobilized on a surface.

Main Results:

Successfully applied the assay to quantify mRNA levels of Klf4, Sox5, and Gapdh in K562 cells.
Demonstrated accurate and parallel measurement of multiple gene transcripts.
Observed a significant downregulation of Sox5 mRNA levels following phorbol 12-myristate 13-acetate induction.
Validated the sensitivity and accuracy of the mRNA mediate-ligation and BEAMing technique.

Conclusions:

The mRNA mediate-ligation and BEAMing technique provides a sensitive and accurate method for quantifying multiple specific mRNAs.
This assay is valuable for studies requiring precise parallel mRNA quantification in small cell populations.
The method facilitates gene expression analysis in contexts with limited cellular material, such as primary cells or sorted populations.