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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: Feb 25, 2026

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

Published on: May 25, 2015

Technical aspects and recommendations for single-cell qPCR.

Anders Ståhlberg¹, Mikael Kubista²

¹Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Box 425, 40530 Gothenburg, Sweden.

Molecular Aspects of Medicine

|July 31, 2017

Summary

This summary is machine-generated.

Quantitative PCR (qPCR) enables molecular profiling of single cells, crucial for understanding cell function and interaction. This guide details the workflow, aiming to standardize single-cell qPCR for broader research application.

Keywords:

MIQE guidelines Single-cell analysis Single-cell qPCR Single-cell workflow qPCR

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Area of Science:

Molecular Biology
Cell Biology
Genomics

Background:

Single cells are fundamental biological units with diverse functions in tissues and organs.
Molecular profiling of single cells is essential for distinguishing cell types, understanding functions, and interactions.
Various single-cell profiling technologies have emerged, yielding significant discoveries over the past decade.

Purpose of the Study:

To discuss technical aspects and provide recommendations for single-cell quantitative PCR (qPCR) analysis.
To highlight the potential of single-cell qPCR as a routine methodology.
To promote standardization and broader adoption of single-cell qPCR techniques.

Main Methods:

Detailed discussion of the single-cell qPCR workflow, encompassing experimental design, sample preparation, and single-cell collection.
Explanation of key steps including direct lysis, reverse transcription, preamplification, and qPCR.
Emphasis on data analysis, detailed reporting, and data sharing for validation and development.

Main Results:

Single-cell qPCR allows interrogation of multiple analytes (DNA, RNA, protein) at the individual cell level.
The technique, while challenging due to small molecule handling, offers significant potential for detailed cellular analysis.
Standardization efforts are crucial for advancing single-cell qPCR from specialized labs to routine research settings.

Conclusions:

Single-cell qPCR is a powerful method for molecular profiling, aiding in the characterization of individual cells.
Addressing technical challenges and promoting standardization will facilitate the routine use of single-cell qPCR.
Widespread adoption of standardized single-cell qPCR will enhance our understanding of cellular heterogeneity and function.