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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

PCR

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

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LEM-PCR: a method for determining relative transcript isoform proportions using real-time PCR without a standard

S Virtue¹, M Dale, J K Sethi

¹Institute of Metabolic Science, Metabolic Research Laboratories, University of Cambridge, Box 289, Level 4, Addenbrooke's Hospital, Cambridge, UK. sv234@medschl.cam.ac.uk

|August 21, 2010

Summary

This study introduces a novel method to accurately quantify messenger RNA (mRNA) isoform proportions in cDNA samples. The new titration curve approach overcomes limitations of traditional standard curves for precise isoform abundance analysis.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Alternative splicing generates multiple messenger RNA (mRNA) isoforms from a single gene.
Accurate quantification of individual mRNA isoform proportions is crucial for understanding gene regulation.
Existing methods using standard curves with recombinant or genomic DNA can lead to inaccurate isoform abundance estimations due to differing amplification efficiencies.

Purpose of the Study:

To develop a more accurate method for determining the relative proportions of different mRNA isoforms within a cDNA sample.
To overcome the limitations of traditional standard curve methods in quantifying isoform abundance.

Main Methods:

The study utilizes a titration curve generated directly from the experimental cDNA samples.
This method involves using samples with varying levels of individual isoforms to create the titration curve.
Linear equations are derived from the titration curve data to calculate the precise proportion of each isoform.

Main Results:

The developed titration curve method provides a more accurate determination of mRNA isoform proportions compared to traditional methods.
This approach avoids potential under- or overestimations associated with external DNA standards.
The method allows for reliable quantification of isoform abundances directly within the biological samples.

Conclusions:

The novel titration curve approach offers a significant improvement for accurately measuring mRNA isoform proportions.
This method enhances the reliability of gene expression studies where isoform abundance is critical.
It provides a valuable tool for researchers investigating the functional significance of alternative splicing.