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Related Experiment Videos

Amplification biases: possible differences among deviating gene expressions.

Séverine A Degrelle1, Christelle Hennequet-Antier, Hélène Chiapello

  • 1Biologie du Développement et Reproduction UMR 1198; ENVA; CNRS, FRE 2857, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France. severine.degrelle@jouy.inra.fr

BMC Genomics
|January 30, 2008
PubMed
Summary
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Moderate RNA amplification using PCR or IVT can introduce biases in gene expression profiling, affecting 16% of probes. However, differential expression analysis remains reliable for biological insights, especially with custom arrays.

Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Gene expression profiling is crucial for studying disease and development.
  • Limited sample material often necessitates RNA amplification.
  • Standard amplification methods include in vitro transcription (IVT) and polymerase chain reaction (PCR).

Purpose of the Study:

  • To investigate biases introduced by moderate RNA amplification (PCR and IVT) in gene expression profiling.
  • To assess the impact of amplification on sequence features and transcript abundance.
  • To evaluate the reliability of differential expression analysis post-amplification.

Main Methods:

  • Screening a bovine cDNA array with embryonic and somatic tissues.
  • Performing moderate amplification (1 round) using global PCR or IVT from 1 µg total RNA.

Related Experiment Videos

  • Analyzing gene expression deviations based on sequence characteristics and transcript levels.

    • Main Results:

    • Moderate amplification affected 16% of probes, causing deviations in gene expression.
    • Biases were linked to sequence molecular features and transcript abundance.
    • Differential expression analysis between embryonic stages yielded reliable and biologically validated results.
    • Relative expression levels were similar between amplified and unamplified samples.

    Conclusions:

    • Systematic biases occur even with moderate PCR and IVT amplification, irrespective of sample type, amplification method, or protocol optimization.
    • Custom, well-suited arrays enhance the search for differential gene expressions.
    • Moderate amplification can be a viable strategy when balanced against the need for reliable differential expression analysis.