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Related Concept Videos

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...
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.
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RACE - Rapid Amplification of cDNA Ends02:35

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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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Ribosome Profiling02:24

Ribosome Profiling

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Transcriptome Analysis of Single Cells
07:27

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Published on: April 25, 2011

Efficacy of RNA amplification is dependent on sequence characteristics: implications for gene expression profiling

Nina Duftner1, Jonah Larkins-Ford, Matthieu Legendre

  • 1Section for Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA.

Genomics
|November 17, 2007
PubMed
Summary

RNA amplification for gene expression profiling can introduce bias. This study found minor amplification bias in cichlid fish RNA, influenced by sequence-specific properties, recommending pre-experiment bias control.

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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
11:52

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Gene expression profiling often utilizes minute samples, necessitating RNA amplification.
  • Current amplification protocols may introduce bias, altering RNA template abundance.
  • Understanding and mitigating this bias is crucial for accurate expression analysis.

Purpose of the Study:

  • To evaluate the amplification bias introduced by a T7 polymerase-based RNA amplification technique.
  • To identify sequence-specific properties that influence RNA amplification bias.
  • To assess the impact of amplification on gene expression data from cichlid fish tissues.

Main Methods:

  • RNA amplification using a T7 polymerase-based method on cichlid fish tissues.
  • Comparison of gene expression levels between unamplified and amplified RNA using cDNA microarrays.
  • Analysis of sequence-specific properties (e.g., GC content, folding energy) affecting amplification.

Main Results:

  • Generally minor amplification bias was observed, with small percentages of lost (1.3%) or gained (2.5%) features.
  • A notable proportion of features were inconsistently regulated before and after amplification (4.2% vs. 19.5%).
  • GC content, folding energy, hairpin characteristics, and poly(A)/poly(T) stretch lengths significantly impacted amplification bias.

Conclusions:

  • T7 polymerase-based RNA amplification can introduce quantifiable bias in gene expression studies.
  • Sequence-specific RNA features are critical determinants of amplification bias.
  • Preceding experiments to control for amplification bias are recommended when using amplified RNA for gene expression analysis.