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Related Concept Videos

Real Time RT-PCR02:57

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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Related Experiment Video

Updated: Jul 15, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
07:27

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Multiplexed RT- PCR for high throughput screening applications.

Derrick Maley1, Jay Mei, Hong Lu

  • 1Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA 19477, USA.

Combinatorial Chemistry & High Throughput Screening
|December 8, 2004
PubMed
Summary

We developed a streamlined TaqMan RT-PCR method for high-throughput gene expression profiling. This single-tube assay enhances efficiency for quantitative mRNA analysis and compound testing in cell cultures.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • High-throughput gene expression profiling is crucial for biological research and drug discovery.
  • Existing methods can be time-consuming and labor-intensive, limiting efficiency.
  • Quantitative mRNA analysis is essential for understanding cellular responses and disease mechanisms.

Purpose of the Study:

  • To establish an efficient, high-throughput TaqMan RT-PCR method for quantitative mRNA expression analysis.
  • To validate a multiplexed, single-tube assay for cell culture applications, including compound testing.
  • To optimize the method for detecting mRNA signals from varying cell numbers.

Main Methods:

  • Development of a novel TaqMan RT-PCR protocol integrating RNA extraction, reverse transcription (RT), and PCR in a single tube.
  • Utilization of poly-A mRNA capture plates for efficient RNA isolation.
  • Validation of multiplexed assays using VIC- and FAM-labeled probes for simultaneous detection of multiple mRNA targets.
  • Optimization of reaction conditions, including cell input for mRNA detection.

Main Results:

  • Demonstrated a linear response for GAPDH mRNA detection across a wide range of cell concentrations (10,000 to 10 cells).
  • Successfully multiplexed different mRNA targets in single PCR reactions without compromising RT-PCR efficiency.
  • Validated the utility of the method for compound screening applications, measuring gene induction.
  • Showcased potential for accelerating target identification, biomarker discovery, and ADMET toxicity assessments.

Conclusions:

  • The novel single-tube TaqMan RT-PCR method significantly enhances throughput for quantitative mRNA analysis.
  • The validated multiplexed assay is effective for cell culture applications, including compound screening and gene induction studies.
  • This technology offers a powerful tool for accelerating various aspects of biological and pharmaceutical research.