Microarray analysis techniques: Difference between revisions

Revision as of 01:45, 3 January 2008

Revision as of 23:24, 13 January 2008

Line 18:

==Aggregation and normalization==

Comparing two different arrays, or two different samples hybridized to the same array generally involves making adjustments for systematic errors introduced by differences in procedures and dye intensity effects. Dye normaization for two color arrays is often acheivedachieved by [[local regression]]. LIMMA provides a set of tools for background correction and scalling, as well an option to average on-slide duplicate spots.<ref>{{cite web |url=http://bioinf.wehi.edu.au/limma/ |title=LIMMA Library: Linear Models for Microarray Data |accessdate=2008-01-01 |format= |work=}}</ref>

Raw Affy data contains about twenty probes for the same RNA target. Half of these are "mismatch spots", which do not precisely match the target sequence. These can theoretically measure the amount of nonspecific binding for a given target. RMA is ana normalization approach that does not take advantage of them, but still must summarize the perfect matches through [[median polish]].<ref>{{cite journal |author=Bolstad BM, Irizarry RA, Astrand M, Speed TP |title=A comparison of normalization methods for high density oligonucleotide array data based on variance and bias |journal=Bioinformatics |volume=19 |issue=2 |pages=185–93 |year=2003 |pmid=12538238 |doi=}}</ref>. Quantile normalization, also part of RMA, is one sensible approach to normalize a batch of arrays in order to make further comparisons meaningful.

The current Affymetrix MAS5 algorithm, which uses both perfect match and mismatch probes, continues to enjoy popularity and do well in head to head tests.<ref>{{cite journal |author=Lim WK, Wang K, Lefebvre C, Califano A |title=Comparative analysis of microarray normalization procedures: effects on reverse engineering gene networks |journal=Bioinformatics |volume=23 |issue=13 |pages=i282–8 |year=2007 |pmid=17646307 |doi=10.1093/bioinformatics/btm201}}</ref>

Line 26:

==Identification of significant differential expression==

Many strategies exist to idenitfy which array probes show an unusual level of over expression or under expression. The simplest one is to call "significant" any probe that differs by an average of at least two fold between treatment groups. More sophicatedsophisticated approaches are often related to [[t test]]s or other mechanisms that take both effect size and variability into account. Curiously, the p-values associated with particular genes do not reproduce well between replicate experiments, and lists generated by straight fold change perform much better.<ref>{{cite journal |author=Shi L, Reid LH, Jones WD, ''et al'' |title=The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements |journal=Nat. Biotechnol. |volume=24 |issue=9 |pages=1151–61 |year=2006 |pmid=16964229 |doi=10.1038/nbt1239}}</ref><ref>{{cite journal |author=Guo L, Lobenhofer EK, Wang C, ''et al'' |title=Rat toxicogenomic study reveals analytical consistency across microarray platforms |journal=Nat. Biotechnol. |volume=24 |issue=9 |pages=1162–9 |year=2006 |pmid=17061323 |doi=}}</ref>This represents an extremely important observation, since the point of performing experiments has to do with predicting general behavior. The MAQC group recommends using a fold change assesmentassessment plus a non-stringent p-value cuttoff, further pointing out that changes in the background correction and scaling proess have only a minimal impact on the rank order of fold change differences, but a substantial impact on p values.

==Pattern recognition==

Commercial systems for gene network analysis such as Ingenuity <ref> {{cite web |url=http://www.ingenuity.com/ |title=Ingenuity Systems |accessdate=2007-12-31 |format= |work=}} </ref> and Pathway studio<ref>{{cite web |url=http://www.ariadnegenomics.com/products/pathway-studio/ |title=Ariadne Genomics: Pathway Studio |accessdate=2007-12-31 |format= |work=}}</ref> create visual representations of differentially expressed genes based on current scientific literature. Non-commercial tools such as [[GenMAPP]] also aid in organizing and visualizing gene network data procured from one or several microarray experiments. A wide variety of microarray analysis tools are availibleavailable through [[Bioconductor]] written in the [[R programming language]]. The frequently cited SAM Excel module and other microarray tools <ref>[{{cite web |url=http://www-stat.stanford.edu/~tibs/SAM/ |title=Significance Analysis of Microarrays |accessdate=2007-12-31 |format= |work=}}]</ref> are available through Stanford University. Another set is available from Harvard and MIT<ref>{{cite web |url=http://www.broad.mit.edu/tools/software.html |title=Software - Broad |accessdate=2007-12-31 |format= |work=}}</ref>

Specialized software tools for statistical analysis to determine the extent of over- or under-expression of a gene in a microarray experiment relative to a reference state have also been developed to aid in identifying genes or gene sets associated with particular [[phenotype]]s. One such method of analysis, known as Gene Set Enrichment Analysis (GSEA), uses a [[Kolmogorov-Smirnov]]-style statistic to identify groups of genes that are regulated together <ref>{{cite journal |author=Subramanian A, Tamayo P, Mootha VK, ''et al'' |title=Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=102 |issue=43 |pages=15545–50 |year=2005 |pmid=16199517 |doi=10.1073/pnas.0506580102}}</ref>. This third-party statistics package offers the user information on the genes or gene sets of interest, including links to entries in databases such as NCBI's [[GenBank]] and curated databases such as Biocarta <ref>{{cite web |url=http://www.biocarta.com/ |title=BioCarta - Charting Pathways of Life |accessdate=2007-12-31 |format= |work=}}] </ref> and [[Gene Ontology]]. Related system, PAINT<ref>{{cite web |url=http://www.dbi.tju.edu/dbi/staticpages.php?page=tools&menu=37 |title=DBI Web |accessdate=2007-12-31 |format= |work=}}}</ref> and SCOPE <ref>{{cite web |url=http://genie.dartmouth.edu/scope/ |title=SCOPE |accessdate=2007-12-31 |format= |work=}}</ref>performs a statistical analysis on gene promoter regions, identifying over and under representation of previously identified [[transcription factor]] response elements.