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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: Jun 8, 2026

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays
10:58

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Published on: December 3, 2010

An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA.

Céline Jeanty1, Dan Longrois, Paul-Michel Mertes

  • 1Laboratory of Cardiovascular Research, Centre de Recherche Public-Santé, Luxembourg.

BMC Genomics
|October 9, 2010
PubMed
Summary
This summary is machine-generated.

Optimizing the reverse transcription (RT) protocol allows for quantitative real-time PCR (qPCR) validation of microarray data using amplified amino allyl labeled RNA (AA-aRNA). This method is crucial when limited RNA is available, improving gene expression analysis.

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Last Updated: Jun 8, 2026

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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

Area of Science:

  • Molecular Biology
  • Genomics
  • Biomarker Discovery

Background:

  • Quantitative real-time PCR (qPCR) validation of microarray data is often hindered by limited RNA quantity.
  • Amplified amino allyl labeled RNA (AA-aRNA) from microarrays presents a potential source for validation experiments.
  • The study investigates the feasibility of using AA-aRNA for qPCR validation in the context of identifying graft rejection biomarkers.

Purpose of the Study:

  • To develop and validate an optimized reverse transcription (RT) protocol for quantitative real-time PCR (qPCR) analysis of amplified amino allyl labeled RNA (AA-aRNA).
  • To enable reliable validation of microarray data using limited RNA samples, particularly in biomarker discovery studies.

Main Methods:

  • Modified the reverse transcription (RT) protocol for AA-aRNA, including a denaturation step, room temperature incubation, transcription initiation enhancement, and RNase H treatment.
  • Tested the optimized RT protocol on universal reference AA-aRNA and AA-aRNA from brain-dead organ donors.
  • Validated the method by comparing qPCR results with microarray data.

Main Results:

  • The optimized RT protocol significantly improved qPCR performance on AA-aRNA, increasing Cq values by an average of 3.4 cycles and enhancing qPCR efficiency.
  • The modifications enabled the detection of previously undetectable low-abundant genes.
  • qPCR results obtained using the optimized protocol showed a significant correlation with microarray data.
  • The method demonstrated a gain of 2.7 cycles on AA-aRNA from 15 donors.

Conclusions:

  • An optimized RT protocol for validating microarray data by qPCR using AA-aRNA has been successfully developed.
  • This method is particularly valuable for gene expression analysis in experiments with limited RNA availability.
  • The findings facilitate more robust validation of transcriptomic data, especially in biomarker discovery research.