Affymetrix Gene Expression

More Information

Latest information on Affymetrix products my be located at the Affymetrix web site.

NOTE: The Partners HealthCare Center for Personalized Genetic Medicine is not affiliated with Affymetrix®. The link to these pages is provided solely as a convenience to PCPGM customers and in no way implies an endorsement of Affymetrix® or any of its products or services by Partners ® HealthCare or vice versa.

Affymetrix arrays are now available in versions using 3’ labeling methods and a newer design for whole transcriptome analysis. Affymetrix single cartridge arrays are used one sample per array. Arrays are manufactured for a wide variety of species. We are able to process cartridge arrays but do not support the plate arrays at this time.

 

Full Service

We start with your total RNA and perform all steps of labeling, hybridization and scanning. Each RNA sample submitted must have the minimums indicated below. Please note that we do not perform concentration of more dilute RNA samples submitted to us. All samples are subjected to QC prior to labeling. See Agilent 2100 Bioanalyzer for QC information.

Standard mass amount of total RNA = 200ng contained in 30-50ng/ul plus a separate tube with an additional 3ul for QC. Samples should have 260/280 = 1.75-2.0 and pass a RIN score of 7 or better. If you have more limiting amounts or RNA, please contact us to discuss possible options.

Samples are labeled according to methods for NuGEN reagent, Ambion, or Affymetrix GeneChip 3' IVT Express™. Methods are selected based on discussions with clients and individual project requirements.

Some RNA extraction kits for micro amounts of material indicate using a polyA carrier to help increase RNA recovery during the preps. USE OF THIS polyA CARRIER SHOULD BE AVOIDED AND ANOTHER OPTION SUBSTITUTED IN ITS PLACE. Reagents used in microrarray preparation will also amplify these carrier molecules and interfere with the analysis.

 

Hybridize/Scan Only

We may receive your samples as is in ready to hybridize form and without further QC on our part and we then perform all steps of hybridization and scanning returning the data files to you. These samples will be hybridized according to the appropriate chip protocol and then scanned according to Affymetrix protocols.

 

Analysis/Bioinformatics with Full Service

Associated with our “Full Service” options we provide a standard analysis report described below. You may also wish to use parts of these descriptions in conjunction with any publications of data. Analysis reports and data may be downloaded from the LIMS. Please review these instructions if you need assistance.

The PCPGM Microarray team performs standard validation procedures for the quality of RNA samples, labeling, and hybridization for all microarray studies. Our microarray instruments are on service contract and subject to routine PM maintenance under those contracts. In addition, there is an examination of the data output from either Affymetrix or Illumina expression analysis platforms. Before any analysis is performed the data are normalized based on results from our examination of the output and QC process. As part of our standard analysis report with our Full Service option we do background correction, average normalization, and compare control vs. experimental sets. We provide fold-change and p-values for each of the differentially expressed probes with an experimental set. This is obtained for each comparison made between groups of samples.

We provide fold-change and a score for each of the differentially expressed probe sets. The top 200 up- and down-regulated genes are separated and presented as an html file with suffix “selected”. A heat-map with probes linking to Affy site (http://www.affymetrix.com/estore/) is also generated for each comparison made.

 

Services specifically associated with Affymetrix Exon 1.0 ST Arrays

Provision of our standard Full Service report for Exon arrays includes use of exonmap and xmapcore R packages for analyzing Affymetrix Exon Array data. Briefly, we do RMA normalization on intensity values. We then provide, for those probesets with magnitude fold-change >1 and p-values <10-4, a mapping to genes. The p-value threshold may be adjusted in order to obtain a reasonable number of top hits (e.g., greater than 100). Please Note: More complex types of analysis such as investigating splicing variation within a limited number of genes is possible. Any analysis for Exon arrays beyond the standard level must be discussed with us in advance of any samples being sent to us.

Projects submitted under “Full Service” will have their data analysis performed and a primary analysis package returned to their GIGPAD account data page in the standard report formats as described.

 

More advanced analysis

We do not have an extensive bioinformatics staff and additional analysis support beyond our standard report will only be undertaken after we agree that the resources available to be devoted to the work are suitable for the requested scope of analysis. More complex types of analysis such as time-series analysis, Gene Set Enrichment Analysis (GSEA), customized heat maps, pathway analysis, and additional simple analysis plots using R may be possible but we consider this sort of analysis beyond the scope of our "Full Service" and these "advanced" services would be at an additional charge over the base service cost. If you believe you will require this more advanced level of support, please discuss this need with us in advance of sending any samples for analysis. We need to determine if the experimental design and statistical power of the replicates provided would be adequate to justify the use of more advanced tools. Requests that involve the use of unusual software or algorithms most often require more time than we are able to dedicate.

NOTE: In both Affymetrix and Illumina analysis, comparison between groups can be made with statistical significance ONLY when each group has at least three biological replicates. If less that three replicates are used statistical significance cannot be established and the customer has to primarily use fold-change to obtain differentially expressed genes.

  1. 1. Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP: GenePattern 2.0, Nat Genet 2006, 38:500-501