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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 13, 2026

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Published on: October 9, 2016

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts.

Keren B Turton¹, Stephane Esnault², Larissa P Delain²

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison; turton@wisc.edu.

Journal of Visualized Experiments : Jove

|October 22, 2016

Summary

This summary is machine-generated.

Researchers developed a quantitative PCR method to measure STAT3 splice variants, revealing co-splicing interdependence and adaptable for other tandem splicing sites.

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Last Updated: Mar 13, 2026

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

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Using the E1A Minigene Tool to Study mRNA Splicing Changes

Using the E1A Minigene Tool to Study mRNA Splicing Changes

Published on: April 22, 2021

Area of Science:

Molecular Biology
Gene Expression Regulation
RNA Splicing

Background:

Signal transducer and activator of transcription 3 (STAT3) gene exhibits complex alternative splicing.
Two distinct splicing events occur: inclusion/exclusion of Serine-701 (S/ΔS) and alternative transactivation domain termini (α/β).
Accurate quantification of these STAT3 splice variants is crucial for understanding gene regulation.

Purpose of the Study:

To develop and validate a quantitative polymerase chain reaction (qPCR) protocol for precise measurement of STAT3 splice variants.
To investigate the fluctuations of STAT3 splice variant levels in response to cytokine treatment in eosinophils.
To explore potential co-splicing interdependence between the two STAT3 splicing events.

Main Methods:

Absolute qPCR using calibrator plasmids to specifically distinguish and quantify four STAT3 spliced transcripts (Sα, Sβ, ΔSα, ΔSβ).
Primer validation and optimization of cycling parameters for specificity and efficiency.
Combined absolute and relative qPCR approaches to analyze STAT3 splice variant dynamics and total STAT3 transcripts.

Main Results:

Successfully established a protocol to accurately measure proportions of all four STAT3 spliced variants.
Described dynamic changes in STAT3 splice variant levels in cytokine-treated eosinophils.
Provided evidence for co-splicing interdependence between the two STAT3 splicing events.

Conclusions:

The developed absolute qPCR protocol enables precise quantification of highly similar STAT3 splice variants.
The findings highlight a regulatory co-splicing mechanism in STAT3 gene expression.
This qPCR strategy is adaptable for studying co-splicing events at other tandem splicing sites.