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Related Experiment Video

Updated: Jul 2, 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

Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device.

Jörn Ueberfeld1, Brian McKenna, Ifat Rubin-Bejerano

  • 1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.

Analytical Chemistry
|September 6, 2008
PubMed
Summary

This study introduces a novel quantitative PCR method using reaction mapping and microelectrophoresis. It achieves high-dynamic-range data for multiplex PCR, improving efficiency and reducing costs for gene network analysis.

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Last Updated: Jul 2, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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09:26

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Published on: March 13, 2017

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Quantitative PCR (qPCR) is crucial for gene expression analysis.
  • Multiplex PCR allows simultaneous detection of multiple targets, but faces challenges in dynamic range and accuracy.
  • Existing qPCR methods have limitations in sensitivity and efficiency for complex gene networks.

Purpose of the Study:

  • To develop a high-dynamic-range quantitative PCR method for multiplex reactions.
  • To improve the efficiency and reduce the cost of quantitative gene expression analysis.
  • To overcome limitations of current fluorometric qPCR readout in multiplex assays.

Main Methods:

  • Applied multiple-time-point reaction mapping to generate quantitative data from multiplex PCR.
  • Utilized a multilane microelectrophoresis device with a novel scanning detector for data collection.
  • Implemented double internal calibration using lane standards and gene standards for accurate measurements.
  • Compensated for PCR slope nonidealities across multiplexes.

Main Results:

  • Achieved high-dynamic-range quantitative data (>9 log2 abundance ratio) in multiplex reactions exceeding 20plexes.
  • Reduced PCR cycle number by approximately five cycles compared to commercial fluorometric readout due to sensitive detection.
  • Demonstrated improved per-well efficiency of quantitative expression analysis by a factor of 50-100.
  • Validated scaling properties and sensitivity for 12plex PCR reactions.

Conclusions:

  • The developed reaction mapping approach significantly enhances quantitative PCR for multiplex reactions.
  • This method offers a more sensitive, efficient, and cost-effective alternative to traditional fluorometric qPCR.
  • The approach is highly attractive for the analysis of large gene networks.