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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: Jul 12, 2026

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

New candidate reference measurement procedure for NRAS p.Q61R quantification by digital PCR.

Jessica Petiti¹, Sabrina Caria², Laura Revel¹

¹Division of Advanced Materials Metrology and Life Sciences, Istituto Nazionale di Ricerca Metrologica (INRIM), Turin, Italy.

Methods (San Diego, Calif.)

|July 10, 2026

Summary

A new digital PCR method accurately quantifies the NRAS p.Q61R mutation, crucial for precision oncology. This reference measurement procedure (RMP) improves harmonization and reliability in mutation testing across laboratories.

Keywords:

Digital PCR (dPCR)Molecular testing NRAS p.Q61R Precision oncology Reference Measurement Procedure (RMP)

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Last Updated: Jul 12, 2026

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Published on: May 9, 2019

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Precision oncology relies on accurate somatic mutation quantification for clinical decisions.
Inter-laboratory variability and lack of metrological traceability hinder reliable mutation testing.
Reference Measurement Procedures (RMPs) are vital for standardizing molecular measurements.

Purpose of the Study:

To develop and validate a candidate RMP for NRAS p.Q61R mutation detection and quantification.
To establish metrological traceability for quantitative biomarker assessment.
To improve harmonization and confidence in mutation testing results.

Main Methods:

Development of a digital PCR (dPCR) assay for NRAS p.Q61R.
Systematic optimization for specificity and allele discrimination.
Analytical characterization including linearity, limit of detection, and precision studies.
Measurement uncertainty budget estimation and inter-laboratory assessment.

Main Results:

The dPCR assay demonstrated excellent linearity across a wide variant allele frequency (vAF) range.
Limit of detection was 0.1%, with good repeatability and intermediate precision.
Comparison with a commercial dPCR assay confirmed assay comparability and consistent vAF estimates.
Preliminary assessment supported the transferability and comparability of the candidate RMP.

Conclusions:

A metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification was established.
This RMP can support harmonization of molecular measurements and value assignment of reference materials.
Implementation of the RMP enhances the reliability of quantitative biomarker assessment in precision oncology.