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==Techniques==
Most microarray manufacturers, such as [[Affymetrix]] and [[Agilent]],<ref>{{cite web|url=http://www.chem.agilent.com/Scripts/PCol.asp?lPage=494 |title=Agilent | DNA Microarrays |accessdate=2008-01-02 |format= |work= |deadurl=yes |archiveurl=https://web.archive.org/web/20071222130157/http://www.chem.agilent.com/Scripts/PCol.asp?lPage=494 |archivedate=December 22, 2007 }}</ref> provide commercial data analysis software
===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 normalization for two color arrays is often achieved by [[local regression]]. LIMMA provides a set of tools for background correction and scaling, as well as 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> A common method for evaluating how well normalized an array is, is to plot an [[MA plot]] of the data.▼
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. Robust Multi-array Average (RMA) <ref>{{cite journal|last=Irizarry|first=RA|author2=Hobbs, B |author3=Collin, F |author4=Beazer-Barclay, YD |author5=Antonellis, KJ |author6=Scherf, U |author7= Speed, TP |title=Exploration, normalization, and summaries of high density oligonucleotide array probe level data.|journal=Biostatistics|volume=4|issue=2|pages=249–64|year=2003|pmid=12925520 |doi=10.1093/biostatistics/4.2.249}}</ref> is a normalization approach that does not take advantage of these mismatch spots, but still must summarize the perfect matches through [[median polish]].<ref>{{cite journal |vauthors=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=10.1093/bioinformatics/19.2.185}}</ref> The median polish algorithm, although robust, behaves differently depending on the number of samples analyzed.<ref>{{cite journal |vauthors=Giorgi FM, Bolger AM, Lohse M, Usadel B |title=Algorithm-driven Artifacts in median polish summarization of Microarray data |journal=BMC Bioinformatics |volume=11 |pages=553 |year=2010 |pmid=21070630 |doi=10.1186/1471-2105-11-553 |pmc=2998528}}</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 |vauthors=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>▼
Factor Analysis for Robust Microarray Summarization (FARMS)<ref>{{cite journal | vauthors = Hochreiter S, Clevert DA, Obermayer K | year = 2006 | title = A new summarization method for affymetrix probe level data | url = http://bioinformatics.oxfordjournals.org/cgi/content/short/22/8/943 | journal = Bioinformatics | volume = 22 | issue = 8| pages = 943–949 | doi=10.1093/bioinformatics/btl033 | pmid=16473874}}</ref> is a model-based technique for summarizing array data at perfect match probe level. It is based on a factor analysis model for which a Bayesian maximum a posteriori method optimizes the model parameters under the assumption of Gaussian measurement noise. According to the Affycomp benchmark<ref>http://affycomp.jhsph.edu/</ref> FARMS outperformed all other summarizations methods with respect to sensitivity and specificity.▼
===Identification of significant differential expression===▼
Many strategies exist to identify 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 twofold between treatment groups. More sophisticated 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 |vauthors=Shi L, Reid LH, Jones WD, etal |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 |pmc=3272078}}</ref><ref>{{cite journal |vauthors=Guo L, Lobenhofer EK, Wang C, etal |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=10.1038/nbt1238}}</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 assessment plus a non-stringent p-value cutoff, further pointing out that changes in the background correction and scaling process 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 FunRich,<ref>{{cite web |url=http://www.funrich.org/ |title=FunRich: Functional Enrichment Analysis |accessdate=2014-09-09 |format= |work=}}</ref> [[GenMAPP]] and [[Anduril (workflow engine)#Moksiskaan|Moksiskaan]] also aid in organizing and visualizing gene network data procured from one or several microarray experiments. A wide variety of microarray analysis tools are available 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 |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 |vauthors=Subramanian A, Tamayo P, Mootha VK, etal |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 |pmc=1239896}}</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]]. Protein complex enrichment analysis tool (COMPLEAT) provides similar enrichment analysis at the level of protein complexes.<ref>{{cite journal |vauthors=Vinayagam A, Hu Y, Kulkarni M, Roesel C, etal |title= Protein Complex-Based Analysis Framework for High-Throughput Data Sets. 6, rs5 (2013). |journal= Sci. Signal. |volume=6 |issue=r5 |year=2013 |pmid= 23443684 |doi= 10.1126/scisignal.2003629 |url= http://www.flyrnai.org/compleat/ |pages=rs5 |pmc=3756668}}</ref> The tool can identify the dynamic protein complex regulation under different condition or time points. 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= |deadurl=yes |archiveurl=https://web.archive.org/web/20070705061522/http://www.dbi.tju.edu/dbi/staticpages.php?page=tools |archivedate=2007-07-05 |df= }}</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. Another statistical analysis tool is Rank Sum Statistics for Gene Set Collections (RssGsc), which uses rank sum probability distribution functions to find gene sets that explain experimental data.<ref>{{cite web |url=http://rssgsc.sourceforge.net/ |title=RssGsc |accessdate=2008-10-15 |format= |work=}}</ref> A further approach is contextual meta-analysis, i.e. finding out how a gene cluster responds to a variety of experimental contexts. [[Genevestigator]] is a public tool to perform contextual meta-analysis across contexts such as anatomical parts, stages of development, and response to diseases, chemicals, stresses, and [[neoplasms]].▼
==Tools==
===Significance analysis of microarrays (SAM)===
Significance analysis of microarrays (SAM) is a [[statistics|statistical technique]] for determining whether changes in [[gene expression]] are statistically significant. It was established in 2001 by Virginia Tusher, [[Robert Tibshirani]] and [[Gilbert Chu]], and is distributed in an [[R (programming language)|R-package]] by [[Stanford University]].
SAM identifies statistically significant genes by carrying out gene specific [[Student's t-test|t-tests]] and computes a statistic ''d<sub>j</sub>'' for each gene ''j'', which measures the strength of the relationship between gene expression and a response variable.<ref name="R4"/><ref name="R5"/><ref name="R6"/> This analysis uses [[non-parametric statistics]], since the data may not follow a [[normal distribution]]. The response variable describes and groups the data based on experimental conditions. In this method, repeated [[permutations]] of the data are used to determine if the expression of any gene is significant related to the response. The use of permutation-based analysis accounts for correlations in genes and avoids [[wikt:Special:Search/parametric|parametric]] assumptions about the distribution of individual genes. This is an advantage over other techniques (e.g., [[ANOVA]] and [[Bonferroni correction]]), which assume equal variance and/or independence of genes.<ref name="R7"/>
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===Spot filtering===
Visual identification of local artifacts, such as printing or washing defects, may likewise suggest the removal of individual spots. This can take a substantial amount of time depending on the quality of array manufacture. In addition, some procedures call for the elimination of all spots with an expression value below a certain intensity threshold.
▲==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 normalization for two color arrays is often achieved by [[local regression]]. LIMMA provides a set of tools for background correction and scaling, as well as 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> A common method for evaluating how well normalized an array is, is to plot an [[MA plot]] of the data.
▲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. Robust Multi-array Average (RMA) <ref>{{cite journal|last=Irizarry|first=RA|author2=Hobbs, B |author3=Collin, F |author4=Beazer-Barclay, YD |author5=Antonellis, KJ |author6=Scherf, U |author7= Speed, TP |title=Exploration, normalization, and summaries of high density oligonucleotide array probe level data.|journal=Biostatistics|volume=4|issue=2|pages=249–64|year=2003|pmid=12925520 |doi=10.1093/biostatistics/4.2.249}}</ref> is a normalization approach that does not take advantage of these mismatch spots, but still must summarize the perfect matches through [[median polish]].<ref>{{cite journal |vauthors=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=10.1093/bioinformatics/19.2.185}}</ref> The median polish algorithm, although robust, behaves differently depending on the number of samples analyzed.<ref>{{cite journal |vauthors=Giorgi FM, Bolger AM, Lohse M, Usadel B |title=Algorithm-driven Artifacts in median polish summarization of Microarray data |journal=BMC Bioinformatics |volume=11 |pages=553 |year=2010 |pmid=21070630 |doi=10.1186/1471-2105-11-553 |pmc=2998528}}</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 |vauthors=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>
▲Factor Analysis for Robust Microarray Summarization (FARMS)<ref>{{cite journal | vauthors = Hochreiter S, Clevert DA, Obermayer K | year = 2006 | title = A new summarization method for affymetrix probe level data | url = http://bioinformatics.oxfordjournals.org/cgi/content/short/22/8/943 | journal = Bioinformatics | volume = 22 | issue = 8| pages = 943–949 | doi=10.1093/bioinformatics/btl033 | pmid=16473874}}</ref> is a model-based technique for summarizing array data at perfect match probe level. It is based on a factor analysis model for which a Bayesian maximum a posteriori method optimizes the model parameters under the assumption of Gaussian measurement noise. According to the Affycomp benchmark<ref>http://affycomp.jhsph.edu/</ref> FARMS outperformed all other summarizations methods with respect to sensitivity and specificity.
▲==Identification of significant differential expression==
▲Many strategies exist to identify 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 twofold between treatment groups. More sophisticated 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 |vauthors=Shi L, Reid LH, Jones WD, etal |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 |pmc=3272078}}</ref><ref>{{cite journal |vauthors=Guo L, Lobenhofer EK, Wang C, etal |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=10.1038/nbt1238}}</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 assessment plus a non-stringent p-value cutoff, further pointing out that changes in the background correction and scaling process 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 FunRich,<ref>{{cite web |url=http://www.funrich.org/ |title=FunRich: Functional Enrichment Analysis |accessdate=2014-09-09 |format= |work=}}</ref> [[GenMAPP]] and [[Anduril (workflow engine)#Moksiskaan|Moksiskaan]] also aid in organizing and visualizing gene network data procured from one or several microarray experiments. A wide variety of microarray analysis tools are available 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 |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 |vauthors=Subramanian A, Tamayo P, Mootha VK, etal |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 |pmc=1239896}}</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]]. Protein complex enrichment analysis tool (COMPLEAT) provides similar enrichment analysis at the level of protein complexes.<ref>{{cite journal |vauthors=Vinayagam A, Hu Y, Kulkarni M, Roesel C, etal |title= Protein Complex-Based Analysis Framework for High-Throughput Data Sets. 6, rs5 (2013). |journal= Sci. Signal. |volume=6 |issue=r5 |year=2013 |pmid= 23443684 |doi= 10.1126/scisignal.2003629 |url= http://www.flyrnai.org/compleat/ |pages=rs5 |pmc=3756668}}</ref> The tool can identify the dynamic protein complex regulation under different condition or time points. 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= |deadurl=yes |archiveurl=https://web.archive.org/web/20070705061522/http://www.dbi.tju.edu/dbi/staticpages.php?page=tools |archivedate=2007-07-05 |df= }}</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. Another statistical analysis tool is Rank Sum Statistics for Gene Set Collections (RssGsc), which uses rank sum probability distribution functions to find gene sets that explain experimental data.<ref>{{cite web |url=http://rssgsc.sourceforge.net/ |title=RssGsc |accessdate=2008-10-15 |format= |work=}}</ref> A further approach is contextual meta-analysis, i.e. finding out how a gene cluster responds to a variety of experimental contexts. [[Genevestigator]] is a public tool to perform contextual meta-analysis across contexts such as anatomical parts, stages of development, and response to diseases, chemicals, stresses, and [[neoplasms]].
==See also==
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