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PCR

PCR

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 - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

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Related Experiment Video

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Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Published on: June 20, 2018

Single-channel multiplexing without melting curve analysis in real-time PCR.

Young-Jo Lee¹, Daeyoung Kim¹, Kihoon Lee¹

¹Seegene, Inc., Taewon Bldg., 91 Ogeum-ro, Songpa-Gu, Seoul 138-828, South Korea.

Scientific Reports

|December 16, 2014

Summary

A new real-time PCR method detects multiple DNA targets in one channel without melting curve analysis. This technique enables precise quantification for improved clinical diagnostics and patient care.

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Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

Published on: December 3, 2010

Area of Science:

Biotechnology
Molecular Biology
Analytical Chemistry

Background:

Multiplex real-time PCR is crucial for detecting multiple targets simultaneously.
Current methods often require separate fluorescence channels or melting curve analysis, increasing complexity and cost.
There is a growing demand in the biotechnology industry for simpler, more efficient multiplexing techniques.

Purpose of the Study:

To develop a novel analytical real-time PCR technique for multiplex target detection within a single fluorescence channel.
To eliminate the need for melting curve analysis in multiplex real-time PCR.
To enable accurate quantification of multiple targets simultaneously for potential clinical applications.

Main Methods:

Utilized the differential fluorescence signal intensities of targets with distinct melting temperatures (Tm) at specific detection temperatures.
Developed a method to detect a high Tm target independently of a low Tm target based on signal intensity at a chosen temperature.
Identified the low Tm target by analyzing changes in fluorescence signals across two different detection temperatures.
Enabled quantification of each target within the same fluorescence channel.

Main Results:

Successfully demonstrated the detection of multiple targets in a single fluorescence channel.
Achieved target differentiation and quantification without employing melting curve analysis.
The developed technique allows for the detection of a high Tm target irrespective of the presence of a low Tm target.
Quantification of individual targets was feasible within the single channel.

Conclusions:

The novel real-time PCR technique offers a simplified approach to multiplex target detection and quantification.
This method eliminates the requirement for melting curve analysis, reducing experimental complexity.
The ability to quantify multiple targets in a single channel holds promise for advancing clinical diagnostics, particularly in areas like drug treatment monitoring.