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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: Mar 5, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Global preamplification simplifies targeted mRNA quantification.

Thomas Kroneis^1,2, Emma Jonasson¹, Daniel Andersson¹

¹Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.

Scientific Reports

|March 24, 2017

Summary

This summary is machine-generated.

Global preamplification offers a flexible workflow for gene expression profiling in small samples, simplifying analysis. While target-specific preamplification shows higher yield and reproducibility, global methods are sufficient for many applications.

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Last Updated: Mar 5, 2026

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Gene expression profiling is crucial for analyzing biological processes, especially in limited sample quantities like single cells.
Preamplification is necessary to amplify low-abundance RNA molecules for accurate analysis using techniques like quantitative real-time PCR (qPCR).

Purpose of the Study:

To compare the efficiency and reliability of global preamplification versus target-specific preamplification for gene expression analysis.
To evaluate the suitability of global preamplification for analyzing gene expression in small sample sizes and single cells.

Main Methods:

Utilized 96 optimized qPCR assays to study global and target-specific preamplification strategies.
Monitored reactions in real-time using SYBR Green I detection and melting curve analysis.
Compared yield and reproducibility between global and target-specific preamplification using identical total RNA amounts.

Main Results:

Global preamplification yielded 9.3-fold less and showed 1.6-fold lower reproducibility compared to target-specific preamplification.
Despite lower yield, global preamplification performance proved adequate for most downstream applications.
Successfully demonstrated global preamplification's potential by analyzing 15 gene expressions in 60 single cells.

Conclusions:

Global preamplification provides a simplified and flexible workflow for targeted gene expression profiling of small samples.
The study outlines the advantages and disadvantages of global preamplification in comparison to target-specific approaches.
Global preamplification is a viable option for gene expression analysis when sample size is a limiting factor.