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RNA profiling for biomarker discovery: practical considerations for limiting sample sizes.

Danny J Kelly1, Sujoy Ghosh

  • 1Genetics Research, GlaxoSmithKline R&D, Research Triangle Park, NC 27709, USA.

Disease Markers
|March 1, 2005
PubMed
Summary
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Comparing gene expression analysis, low RNA amounts showed poor signal correlation but good fold-change estimates versus standard amounts. Including a reference sample improved signal correlation for low RNA gene expression studies.

Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Microarray analysis is crucial for gene expression profiling.
  • Standard RNA input amounts may not always be feasible.
  • Low RNA input methods require careful validation.

Purpose of the Study:

  • To compare gene expression data from standard and low RNA amounts using Affymetrix microarrays.
  • To evaluate the impact of RNA input quantity on gene signal and fold-change estimates.
  • To assess the utility of reference samples in low RNA input microarray experiments.

Main Methods:

  • Affymetrix microarray hybridization using standard (8 µg) and low (100 ng) total RNA.
  • Analysis of gene signal intensities and fold-change estimations.

Related Experiment Videos

  • Validation of results using real-time polymerase chain reaction (PCR) assays.

    • Main Results:

    • Poor correlation in gene signals between low and standard RNA for low to moderately abundant genes.
    • High abundance genes showed better signal correlation.
    • Inclusion of a reference sample improved signal correlation for low RNA.
    • Fold-change estimates were well-correlated between methods regardless of gene abundance.

    Conclusions:

    • Low RNA input can yield reliable gene fold-change data but may compromise absolute signal accuracy.
    • Reference sample normalization is recommended for comparing gene signals between low and standard RNA inputs.
    • No specific referencing is needed when comparing fold-changes between standard and low RNA template reactions.