Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines...

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• DNA microarrays enable gene expression comparison by adjusting hybridization intensities.
• Global normalization methods rely on all genes or a small subset of modulated genes.
• Alternative normalization strategies are needed for microarrays with unknown gene modulation proportions or biased trends.

Purpose of the Study:

• To investigate the efficacy of spike-in controls for normalizing low-density microarrays.
• To analyze gene modulation in response to hypoxia in a macrophage cell line using microarrays.
• To evaluate spike-in normalization for both symmetric and asymmetric gene expression distributions.

Main Methods:

• Designed a test-array for hypoxia gene modulation analysis in macrophages.
• Extracted RNA from control and hypoxic cells, spiked with bacterial RNAs.
• Hybridized RNA to the test-array containing mouse genes and spike oligonucleotides.

Main Results:

• Demonstrated high reproducibility of the microarray platform.
• Confirmed suitability of spike-in controls for normalization and determining differential gene expression thresholds.
• Found spike-in normalization accurate and comparable to other methods for symmetric data.
• Showed spike-in normalization to be superior and necessary for asymmetric, up-regulated gene expression data.

Conclusions:

• Spike-in control normalization is a reliable and reproducible method.
• This method is advantageous for biased microarray platforms with asymmetric gene expression distributions.
• Applicable to diagnostic chips where gene regulation may be skewed.