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Related Experiment Videos

Reproducibility of oligonucleotide arrays using small samples.

Jeanette N McClintick1, Ronald E Jerome, Charles R Nicholson

  • 1Center for Medical Genomics, Indiana University School of Medicine, Indianapolis, Indiana, USA. jnmcclin@iupui.edu

BMC Genomics
|February 22, 2003
PubMed
Summary

Detecting differential gene expression is feasible with low RNA yields. Standard protocols work with 2 micrograms (µg) of RNA, while amplification allows detection down to 0.1 µg, revealing biological differences.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Low RNA yields from small tissue samples hinder oligonucleotide microarray analysis (Affymetrix GeneChips).
  • Previous methods reducing RNA or amplifying cRNA decreased transcript detection.
  • The impact on detecting biological differences in realistic experiments remained unanalyzed.

Purpose of the Study:

  • To systematically evaluate the effect of varying total RNA amounts on detecting differential gene expression.
  • To assess the feasibility of using reduced RNA quantities in standard and amplified microarray protocols.

Main Methods:

  • Systematic analysis of RNA input amounts for Affymetrix GeneChip experiments.
  • Comparison of standard labeling and hybridization protocols with a single-cycle amplification protocol.

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  • Evaluation of technical versus biological variability in gene expression analysis.

    • Main Results:

    • The standard Affymetrix protocol yields results comparable to the recommended amount when using as little as 2 micrograms (µg) of total RNA.
    • Biological variability significantly outweighs technical variability introduced by reduced RNA amounts.
    • A single-cycle amplification protocol enables detection of differential gene expression from samples as small as 0.1 µg of total RNA, capturing a substantial fraction of significant differences.

    Conclusions:

    • Standard protocols are effective with 2 µg RNA; modifications may allow use of 1 µg.
    • Differential gene expression detection is achievable with as little as 0.1 µg RNA using amplification.
    • Biological variations are more substantial than technical variations from RNA manipulation; larger sample group comparisons (four arrays) yield more significant differences.