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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...
DNA Microarrays02:34

DNA Microarrays

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...

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Related Experiment Video

Updated: May 24, 2026

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
10:50

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Published on: February 25, 2017

A novel procedure for absolute real-time quantification of gene expression patterns.

Yingqing Lu1, Lulu Xie, Jiani Chen

  • 1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nan Xin Cun, Beijing 100093, China. yqlu@ibcas.ac.cn.

Plant Methods
|March 13, 2012
PubMed
Summary

A novel cDNA-based absolute quantitative polymerase chain reaction (qPCR) method accurately estimates absolute transcript numbers. This technique enhances sensitivity to gene expression variations, enabling cross-experiment comparisons for diverse biological systems.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Gene expression patterns are crucial for biological system function.
  • Real-time quantitative polymerase chain reaction (qPCR) is widely used but often limited to relative quantification.
  • Absolute transcript quantification methods face unresolved challenges.

Purpose of the Study:

  • To introduce a novel procedure for accurate absolute quantification of transcripts.
  • To overcome limitations of current qPCR methods for gene expression analysis.

Main Methods:

  • Development of a cDNA-based absolute (CBA) qPCR procedure.
  • Quantification of total cDNA at a resolution of approximately 70 pg/μl.
  • Estimation of absolute transcript numbers per picogram of cDNA.

Main Results:

  • The CBA qPCR method accurately quantifies absolute transcript numbers.
  • CBA qPCR is more sensitive to gene expression variations (developmental, environmental) than relative quantification.
  • Normalization by reference transcripts allows detection of cell-level expression patterns across species and tissues.

Conclusions:

  • The new procedure enables direct comparison of gene expression results across experiments.
  • Facilitates inferences of gene expression patterns in diverse living systems and tissues.
  • Addresses the need for robust biological interpretations of gene expression variations.