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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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...
67.1K

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Related Experiment Video

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A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations
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A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR.

Hao Sun1, Tim Olsen2, Jing Zhu2

  • 1Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China ; Department of Mechanical Engineering, Columbia University, New York, NY, USA.

RSC Advances
|April 18, 2015
PubMed
Summary

This study introduces an integrated microfluidic device for single-cell gene expression analysis using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The method simplifies the process, minimizes sample loss, and enables efficient analysis of gene induction in individual cells.

Keywords:
MicrobeadMicrofluidicRT-qPCRSingle cell analysis

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Genomics

Background:

  • Single-cell gene expression analysis is crucial for understanding cellular heterogeneity.
  • Existing methods for single-cell gene expression analysis can be complex and prone to sample loss.
  • Microfluidic platforms offer potential for streamlined and efficient single-cell assays.

Purpose of the Study:

  • To develop an integrated microfluidic approach for single-cell reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).
  • To simplify the multi-step process of gene expression analysis from a single cell.
  • To demonstrate the utility of the developed platform for studying drug-induced gene expression changes.

Main Methods:

  • A microfluidic device was designed to integrate cell isolation, lysis, messenger RNA (mRNA) purification, reverse transcription, and quantitative real-time PCR (qPCR).
  • Reactions were performed on microbeads within a single microchamber for simplified operation and reduced sample loss.
  • Temperature control was achieved using an integrated heater and temperature sensor for precise reaction conditions.

Main Results:

  • The microfluidic approach successfully integrated all functional steps for single-cell RT-qPCR.
  • The use of microbeads simplified device design and operation, minimizing sample loss and contamination.
  • The platform demonstrated its utility by analyzing the drug-induced expression of CDKN1A in single human cancer cells (MCF-7).

Conclusions:

  • The developed microfluidic device provides an efficient and integrated solution for single-cell gene expression analysis via RT-qPCR.
  • This approach facilitates the study of gene expression variations at the single-cell level.
  • The platform holds potential for applications in drug screening and understanding cellular responses to stimuli.