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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...

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

PCR

PCR

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pcrEfficiency: a Web tool for PCR amplification efficiency prediction.

Izaskun Mallona¹, Julia Weiss, Marcos Egea-Cortines

¹Genetics, Institute of Plant Biotechnology (IBV), Technical University of Cartagena (UPCT), Campus Muralla del Mar, 30202 Cartagena, Spain. izaskun.mallona@upct.es

BMC Bioinformatics

|October 22, 2011

Summary

This summary is machine-generated.

Predicting PCR efficiency is crucial for accurate gene expression analysis. A new web tool, pcrEfficiency, uses a statistical model to forecast amplification efficiency, improving quantitative PCR experimental design.

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Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

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Area of Science:

Molecular Biology
Bioinformatics

Background:

Accurate gene expression analysis using quantitative PCR (qPCR) relies on comparing amplification efficiencies between reference and target genes.
Discrepancies in PCR efficiency can lead to significant miscalculations in gene expression levels.
Existing primer design tools do not predict PCR efficiency.

Purpose of the Study:

To develop a method for predicting PCR amplification efficiency for primer pairs and amplicons.
To create a user-friendly tool for optimizing qPCR experiments.

Main Methods:

A statistical approach using a generalized additive model was employed.
Data from 90 primer pair combinations across diverse organisms (bacteria, yeast, plants, humans) and amplicon sizes (74-907 bp) were analyzed.
An open-source web interface was developed.

Main Results:

Key parameters influencing PCR efficiency were identified.
The developed model accurately predicts amplification efficiencies.
The web interface allows users to obtain optimized oligonucleotides with predicted PCR efficiencies.

Conclusions:

The pcrEfficiency web tool simplifies qPCR setup by enabling pre-laboratory prediction of PCR efficiencies.
This resource facilitates more reliable gene expression analysis.
The tool and its source code are freely available under the GPL v2 license.