Illumina Gene Expression

More Information

If you wish to review more information about these arrays please refer to the Illumina web site. For content information on these array types, visit the Illumina web site page containing the gene lists and download for the format of interest.

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

Illumina BeadChip arrays are arranged in array of arrays format providing configurations for six, eight or 12 individual arrays on each BeadChip array. The arrays are separated from one another by a seal so that each array can be hybridized to a different sample; and multiple samples may be interrogated simultaneously. While not limiting, the most efficient use of these arrays is to plan for sample numbers and arrays that allow us to fill the greatest percentage of slots on each single BeadChip array.

These arrays are available for human, mouse and rat samples. According to Illumina WG-6 and HT-12 arrays provide genome-wide transcriptional coverage of well-characterized genes, gene candidates, and splice variants, with a significant portion targeting well-established sequences supported by peer-reviewed literature.

 

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. Please see Agilent 2100 Bioanalyzer section 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 may be labeled using the NuGEN reagents or Ambion Illumina TotalPrep RNA Amplification kits. Methods are selected based on discussions with clients and individual project requirements.

NOTE: Some RNA extraction kits used for micro amounts of material supply a polyA carrier to 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 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 Illumina protocols. Please ensure you inform the lab if you have used any Nugen reagents, as this has implications on the hybridization method. Chips offered: Samples of Human origin: Illumina Human WG6 v3, Illumina HT-12, Illumina Ref 8. Samples of Mouse origin: Illumina WG 6 v2, Illumina Ref 8."

 

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. For Illumina array results analysis we use GenomeStudio (Illumina, Inc.) to perform the analysis. Briefly we do 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.

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.

We are unable to offer any GenomeStudio licenses for customer use, please contact Illumina (www.illumina.com) for more information on purchasing GenomeStudio. Alternative software suggestions are R Bioconductor package (http://www.bioconductor.org/) or GenePattern (http://www.broadinstitute.org/cancer/software/genepattern/). These suggestions are not endorsements of these products."

 

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.