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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: Apr 18, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Published on: January 8, 2008

Pyrosequencing for accurate imprinted allele expression analysis.

Bing Yang¹, Nathan Damaschke¹, Tianyu Yao¹

¹Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.

Journal of Cellular Biochemistry

|January 13, 2015

Summary

This summary is machine-generated.

We developed Pyrosequencing for Imprinted Expression (PIE), a rapid assay to measure gene expression from specific parental alleles. This method accurately quantifies imprinting and heterozygosity, aiding in understanding related diseases.

Keywords:

EPIGENETIC IGF2 IMPRINTING INTRON-CROSSING PRIMER PYROSEQUENCING

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Pyrosequencing: A Simple Method for Accurate Genotyping

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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Published on: February 10, 2023

Area of Science:

Epigenetics
Molecular Biology
Genetics

Background:

Genomic imprinting regulates gene expression, with errors linked to diseases.
Accurate quantification of parental allele contribution is crucial for imprinting studies.
Existing methods have limitations in speed, labor, and accuracy.

Purpose of the Study:

To develop a rapid and sensitive quantitative assay for measuring individual allelic ratios.
To overcome limitations of current methods for assessing genomic imprinting.
To apply the assay to study Insulin-like Growth Factor-2 (IGF2) imprinting.

Main Methods:

Development of Pyrosequencing for Imprinted Expression (PIE) assay.
PIE measures individual allelic ratios and genomic heterozygosity.
Application of PIE to human and mouse tissues for IGF2 imprinting analysis.

Main Results:

PIE offers shorter experimental time and reduced labor compared to other methods.
PIE avoids issues like heteroduplex formation seen in qPCR-based assays.
The assay demonstrates high sensitivity, detecting allelic expression differences as low as 1%.

Conclusions:

PIE is a sensitive, rapid, and versatile tool for quantifying allelic expression and imprinting.
The assay simplifies the study of imprinting disorders and gene expression.
PIE successfully determined IGF2 imprinting status in various tissues.