Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile
Varesio, Luigi; Bonassi, Stefano; Ricciardi, Annamaria; Biasotti, Barbara; Moretti, Stefano; Fardin, Paolo (2007), Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile, BMC Genomics, 8, 17. http://dx.doi.org/10.1186/1471-2164-8-17
TypeArticle accepté pour publication ou publié
External document linkhttp://www.biomedcentral.com/1471-2164/8/17
Journal nameBMC Genomics
MetadataShow full item record
Abstract (EN)Background The normalization of DNA microarrays allows comparison among samples by adjusting for individual hybridization intensities. The approaches most commonly used are global normalization methods that are based on the expression of all genes on the slide and on the modulation of a small proportion of genes. Alternative approaches must be developed for microarrays where the proportion of modulated genes and their distribution are unknown and they may be biased towards up- or down-modulated trends. Results The aim of the work is to study the use of spike-in controls to normalize low-density microarrays. Our test-array was designed to analyze gene modulation in response to hypoxia (a condition of low oxygen tension) in a macrophage cell line. RNA was extracted from controls and cells exposed to hypoxia, mixed with spike RNA, labeled and hybridized to our test-array. We used eight bacterial RNAs as source of spikes. The test-array contained the oligonucleotides specific for 178 mouse genes and those specific for the eight spikes. We assessed the quality of the spike signals, the reproducibility of the results and, in general, the nature of the variability. The small values of the coefficients of variation revealed high reproducibility of our platform either in replicated spots or in technical replicates. We demonstrated that the spike-in system was suitable for normalizing our platform and determining the threshold for discriminating the hypoxia modulated genes. We assessed the application of the spike-in normalization method to microarrays in which the distribution of the expression values was symmetric or asymmetric. We found that this system is accurate, reproducible and comparable to other normalization methods when the distribution of the expression values is symmetric. In contrast, we found that the use of the spike-in normalization method is superior and necessary when the distribution of the gene expression is asymmetric and biased towards up-regulated genes. Conclusion We demonstrate that spike-in controls based normalization is a reliable and reproducible method that has the major advantage to be applicable also to biased platform where the distribution of the up- and down-regulated genes is asymmetric as it may occur in diagnostic chips.
Subjects / Keywordsdistribution of the gene expression; macrophage cell line; gene modulation; normalize low-density microarrays; spike-in controls; microarray
Showing items related by title and author.
Identification of low intratumoral gene expression heterogeneity in neuroblastic tumors by genome-wide expression analysis and game theory Tonini, Gian Paolo; Bonassi, Stefano; Stigliani, Sara; Cavazzana, Andrea; Di Cristofano, Claudio; Truini, Mauro; Coco, Simona; Moretti, Stefano; Scaruffi, Paola; Albino, Domenico (2008) Article accepté pour publication ou publié
Genome analysis and gene expression profiling of neuroblastoma and ganglioneuroblastoma reveal differences between neuroblastic and Schwannian stromal cells Cavazzana, Andrea; Di Cristofano, Claudio; Perri, Patrizia; Gambini, Claudio; Longo, Luca; Mazzocco, Katia; Moretti, Stefano; Bonassi, Stefano; Tonini, Gian Paolo; Coco, Simona; Defferrari, Raffaella; Scaruffi, Paola (2005) Article accepté pour publication ou publié
Age-dependent accumulation of genomic aberrations and deregulation of cell cycle and telomerase genes in metastatic neuroblastoma Coco, Simona; Theissen, Jessica; Scaruffi, Paola; Stigliani, Sara; Moretti, Stefano; Oberthuer, André; Valdora, Francesca; Fischer, Matthias; Gallo, Fabio; Hero, Barbara; Bonassi, Stefano; Berthold, Frank; Tonini, Gian Paolo (2012) Article accepté pour publication ou publié
Bone marrow of neuroblastoma patients shows downregulation of CXCL12 expression and presence of IFN signature Parodi, Stefano; Moretti, Stefano; Garaventa, Alberto; Zanazzo, Giulio; Bonassi, Stefano; Fardin, Paolo; Pistoia, Vito; Tonini, Paolo; Corrias, Maria Valeria; Morandi, Fabio; Gallo, Fabio; Scaruffi, Paola; Stigliani, Sara (2012) Article accepté pour publication ou publié
Bone Marrow-Infiltrating Human Neuroblastoma Cells Express High Levels of Calprotectin and HLA-G Proteins Morandi, Fabio; Scaruffi, Paola; Gallo, Fabio; Stigliani, Sara; Moretti, Stefano; Bonassi, Stefano; Gambini, Claudio; Mazzocco, Katia; Fardin, Paolo; Haupt, Riccardo; Pistoia, Vito; Tonini, Gian Paolo; Corrias, Maria Valeria (2012) Article accepté pour publication ou publié