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BioMicroCenter:Microarrays: Difference between revisions
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→QUALITY CONTROL
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== QUALITY CONTROL == | == QUALITY CONTROL == | ||
The BioMicro Center employs several quality controls as part of microarray analysis | |||
=== RNA === | === RNA === | ||
All RNA samples are run on the [[BioMicroCenter:2100BioAnalyzer|Agilent BioAnalyzer]] prior to labeling. The BioAnalyzer results are used to determine the concentration of the RNA and the quality of the RNA based on their [[BioMicroCenter:RIN|RNA Integrity Number (RIN)]] score. | |||
=== Affymetrix Arrays === | === Affymetrix Arrays === | ||
After scanning, 3' Affymetrix arrays are processed through a number of quality control steps to confirm the quality of the array. These steps create quality control plots that we distribute to the user. | |||
==== QC PLOTS ==== | |||
In all cases, the plots are guidelines but not absolutes. Differences in the quality of starting RNA or source of the RNA can have a major impact on the plots. Biological replicates should behave similarly and we focus on changes where we find that is not true. | |||
The RNA Degradation plot measure the relative intensity of probes at the 5' and 3' end of the average gene. Differences in the reverse transcription step can hive significant effects on the outcome. The precise slope of the line is not as important as having the lines have similar slopes. | |||
Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. NUSE scores above 1.05 or disconnected from the others are generally (though not always) considered poor. | |||
The Affy QC pdf contains a number of metrics including box plots and histograms of intensity, QC scores for spike in controls and interarray correlation. | |||
Fianlly, we use gcRMA for normalization. gcRMA normalized data is included in the data we distribute. | |||
==== References ==== | |||
These plots are generated using a number of packages from [http://www.bioconductor.org/ bioconductor] including: | |||
*affy:Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315. | |||
*affyQCReport | |||
*affyPLM: Brettschneider J, Collin F, Bolstad BM, and Speed TP. (2007) Quality assessment for short oligonucleotide arrays. Technometrics. | |||
*LPE: Nitin Jain, Michael O'Connell and Jae K. Lee. Includes R source code contributed by HyungJun Cho <hcho@virginia.edu> (). LPE: Methods for analyzing microarray data using Local Pooled Error (LPE) method. R package version 1.20.0. http://www.r-project.org, | |||
Bioconductor reference: Bioconductor: Open software development for computational biology and bioinformatics R. Gentleman, V. J. Carey, D. M. Bates, B.Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and others 2004, Genome Biology, Vol. 5, R80 | |||
gcRMA: Jean(ZHIJIN) Wu and Rafael Irizarry with contributions from James MacDonald Jeff Gentry (). gcrma: Background Adjustment Using Sequence Information. R package version 2.18.1. | |||
=== Agilent Arrays === | === Agilent Arrays === | ||
- - To Be Added - - | |||
== DATA ANALYSIS == | == DATA ANALYSIS == | ||
