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Related Concept Videos

RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
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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: Jun 17, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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Published on: August 3, 2011

Sensitive multiplex RNA quantification using capillary electrophoresis-based single-strand conformation polymorphism.

Gi Won Shin1, Hee Sung Hwang, Hong Gil Nam

  • 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.

Biotechnology and Bioengineering
|December 17, 2009
PubMed
Summary

This study introduces a novel multiplex RNA quantification method using capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for accurate, high-throughput analysis. The new technique overcomes limitations of real-time PCR, enabling precise quantification of multiple RNA targets simultaneously.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Accurate RNA quantification is vital for biological studies like gene expression analysis.
  • Real-time polymerase chain reaction (PCR) is the standard for RNA quantification but is limited to single targets.
  • Multiplex applications of real-time PCR are hindered by reduced quantification power and dye limitations.

Purpose of the Study:

  • To develop a novel method for multiplex RNA quantification.
  • To overcome the single-target limitation of current real-time PCR methods.
  • To enable robust and precise quantification of multiple RNA targets simultaneously.

Main Methods:

  • Developed a multiplex RNA quantification method combining modified reverse transcription (RT) and asymmetric PCR with capillary electrophoresis single-strand conformation polymorphism (CE-SSCP).
  • RNAs were reverse transcribed, modified with common sequence tags, and amplified using tag-specific primers.
  • Amplicons were separated and quantified using CE-SSCP.

Main Results:

  • The novel CE-SSCP based method demonstrated a limit of detection as low as 2 amol.
  • The assay exhibited a wide dynamic range of approximately 10(5).
  • The method successfully quantified seven in vitro transcribed RNAs in a multiplex format.

Conclusions:

  • The developed CE-SSCP coupled method offers a robust and precise solution for multiplex RNA quantification.
  • This approach overcomes the limitations of single-target analysis inherent in traditional real-time PCR.
  • The method holds significant potential for advancing high-throughput RNA analysis in various biological research fields.