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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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Digital PCR for the characterization of reference materials.

Megan H Cleveland¹, Hua-Jun He¹, Mojca Milavec²

¹Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899, USA.

Molecular Aspects of Medicine

|February 15, 2024

Summary

Digital PCR (dPCR) offers precise absolute quantification for nucleic acid reference materials, surpassing traditional methods. This review details dPCR

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Area of Science:

Biotechnology
Molecular Biology
Metrology

Background:

Nucleic acid-based tests require well-characterized reference materials for accuracy and harmonization.
Digital PCR (dPCR) enables absolute quantification of nucleic acid sequences, making it suitable for reference material characterization.

Purpose of the Study:

To review the technical considerations and applications of digital PCR (dPCR) in developing and characterizing nucleic acid-based reference materials.
To highlight the advantages of dPCR over conventional methods like UV-spectroscopy for reference material certification.

Main Methods:

Review of existing literature and methodologies concerning dPCR application in reference material characterization.
Analysis of dPCR's technical aspects, including optimization, limitations, and advantages.

Main Results:

dPCR provides accurate absolute quantification of nucleic acid targets, crucial for reference material certification.
National Metrology Institutes are increasingly adopting dPCR for reference material characterization.
dPCR offers advantages over indirect methods such as UV-spectroscopy.

Conclusions:

dPCR is a powerful tool for characterizing nucleic acid reference materials, supporting test harmonization and accuracy.
Users must be aware of dPCR's specific requirements and limitations for optimal performance.
The adoption of dPCR signifies a shift towards more precise metrological approaches in molecular diagnostics.