The Micro Array Support Facility
Principal Investigator: M. Alcalay, Milan (IT)
Co-principal Investigator: Heiko Muller, Milan (IT)
The FIRC Institute for Molecular Oncology (IFOM) microarray facility has been operating for the past year and a half on Affymetrix technology. The facility is coordinated by a senior scientist, Myriam Alcalay, is run by two qualified technicians, and currently consists of 2 complete Affymetrix instrumentation systems. The facility performs all aspects of the microarray experiments for its users, including:
1. Counseling on experimental design. In order to obtain reliable results, we routinely advise performing experiments using replicate samples and chips. The number of replicates is dependent on the intrinsic genetic variability of the experimental model system (cell lines or animal models or patient samples), whereas chip performance is stable and a duplicate chip analysis is usually sufficient. The type of chips to be used is selected on the basis of the organism and of the aim of the experiment.
2. Expression profiling experiments, including: biotinylated cRNA synthesis (with the possibility of introducing amplification steps when low quantities of RNA are available), Affymetrix GeneChip hybridization, washing, staining, scanning, and image analysis with Affymetrix Microarray Suite software. We routinely perform data acquisition using both absolute analysis and comparative analysis of test sample versus reference samples. Raw outputs are available to users and are uploaded in a data repository.
3. Data elaboration with tools developed in IFOM and with GeneSpring software. We routinely perform elaboration steps such as replica analysis and application of statistical tests (t-test, ANOVA, McNemar test) to determine data quality and cutoff values. The curated data is uploaded to GeneSpring where global error model normalization is applied. Within this software, specific clustering and filtering of data according to experimental design can also be performed. Fully annotated, relevant gene lists are available to the user for further validation.
The facility currently performs microarray experiments for 15 groups and has a throughput of approximately 80 chips/week. The scientific topics that have been or are being studied using gene expression analysis through our facility include cell cycle regulation (Dr. K. Helin, Dr. H. Muller), myeloid differentiation and acute myeloid leukemogenesis (Dr. M. Alcalay, Prof. P.G. Pelicci), oxidative stress (Prof. P.G. Pelicci), metastasis (Prof. P.P. Di Fiore), thyroid tumors (Prof. M. Pierotti), homeobox gene regulation (Prof. F. Blasi), angiogenesis (Dr. E. Dejana), and others. The IFOM microarray facility also makes use of cDNA arrays for specific studies. The facility is equipped with 2 Molecular Dynamics Lucidea spotters (~ 21000 clones/slide spotting density), 1 Axon 4A scanner (resolution 10µ) and 1 Axon 4B scanner (resolution 5µ). Production of cDNA chips is performed through spotting of PCR products, derived from amplification of plasmid inserts. An important resource for chip production present at IFOM is the complete Sequence Validated Human cDNA Library (Research Genetics), which includes > 40,000 representative clones derived from UniGene clustering. Murine clones and clones not present in this collection can be ordered from IMAGE clone distributors.
Another important technology that is currently being implemented in the IFOM microarray service is ChIP-on-chip, i.e. high throughput analysis of promoter binding by chromatin immunoprecipitation (ChIP). The facility is currently collecting genomic fragments representative of promoter sequences (both in the form of BAC inserts and oligonucleotides). These "promoter arrays" will then be hybridized with fluorescently labeled products deriving from ChIP experiments. The facility intends to produce high-density spotted arrays, representative of all available human and mouse promoters as well as optimizing ChIP assays for transcription factors upon request.
A particular asset of the IFOM microarray facility is the close interaction with the IFOM and the MAIN Consortium's bioinformatics cores, which guarantees constant updating of analysis strategies and of available tools. A lot of effort has been invested in order to coordinate activities and to develop tools that are useful for the elaboration of microarray results. The following tools are available:
· Automated chip annotation tables, visible at http://bio.ifom-firc.it/ARRAY_ANNOT/index.html, permit easy conversion of Affymetrix identifiers to useful, updated gene information
· Chpreader/GenePicker proprietary software package for experimental replica analysis and statistical significance of results, yields highly significant lists of regulated genes
· EST annotation machine and Keyword Clustering program for retrieval and grouping of functional information (http://bio.ifom-firc.it/)
· The Human <> Mouse Promoter Machine (in progress), which will permit batch retrieval of human and/or mouse promoter sequences.
All results currently produced at IFOM are imported to a centralized database (GeNet) for meta-analysis, which is performed solely by IFOM scientists under the supervision of Dr. Heiko Muller. Thus all users can obtain privileged information concerning the behavior of specific genes of interest in other screens, without consulting the other screens themselves. If publication and/or know-how leading to proprietary positions is generated, all of the people whose screenings were utilized will be co-authors or co-inventors. This applies to all data that is not in the public domain (published) at the date of the agreement and does not apply to data that is already in the public domain.