The Microarray & Genomics Facility provides an integrated set of tools and services for high throughput technologies for applied genomics. The Microarray division is funded through the NCI CCSG, RPCI, SUNY Buffalo and chargeback revenues. cDNA and genomic BAC clones, such as those identified in the Genomics division projects, are offered as an arrayed and archived resource. Investigators can request microarray services including RNA/DNA isolation, Cy3 and Cy5 probe generation, hybridizations, custom-made oligonucleotide and genomic arrays, Agilent and NimbleGen arrays, scanning of microarrayed slides, image analysis, normalization of data and web-based interface with an Oracle database. The Genomics division is funded through grants, contracts, and chargeback revenues, and contributes to the application of human and mouse BAC clones for downstream functional analysis. It provides the infrastructure for automation through robotics for large-scale projects. In addition to the cDNA libraries, fifteen BAC/PAC genomic libraries are available for screening, clone selection, characterization, mapping, and distribution. The Genotyping division is funded through the NCI CCSG, RPCI support and chargeback revenues. Investigators can request large scale SNP detection, SNP analysis, copy number determiniation, quantitative gene expression and methylation studies using the Sequenom MassARRAY and Illumina GoldenGate and Infinium systems.

Scheduling

Investigators should contact Jeff Conroy (716-845-3361) for details. Priority is given to NCI-funded investigators with RPCI affiliation, and as determined by the RPCI Microarray Steering Committee.

User Fees

There is a chargeback system for users of the RPCI Microarray and Genomics Facility. The fees vary depending on the affiliation of the investigator, cost of materials and the extent of services requested from the user (see fee schedule). Requisition forms must be completed, including grant/account number and signature, and submitted along with the sample(s). Billing is done at the completion of the service.

Material Transfer Agreements

GENOMIC SERVICES ONLY: Roswell Park Cancer Institute must receive a completed and signed Material Transfer Agreement (Academic MTA, Commercial MTA) from both Academic and Industrial users before RPCI BAC and /or PAC clones can be distributed. Any questions with this agreement should be directed to Dr. Richard Matner, RPCI Tech Transfer (716-845-8192).

Policies

Project Description - Each RPCI investigator is required to submit a brief, one page description of their project, and need for microarray, genotyping or genomic services. Please include program affiliation and grant funding (NCI, etc.). Projects may be reviewed by the Steering Committee before services are performed.

Agilent, NimbleGen and BAC aCGH and gene expression microarray services

Requisitions - All samples, cDNA clones, PCR products, array and hybridization requests must be received with a completed, signed requisition form. Information for clones and PCR products must have a corresponding Excel spreadsheet in a format consistent with the following examples.

Custom arrays - Investigators can select individual oligos, BACs or cDNA clones from the archived clones sets and have them arrayed as custom slides. Investigators are responsible for selecting the oligo-clone name/plate IDs from the database and forwarding the choices to the facility in an Excel spreadsheet and completed microarray fabrication requisition (EXAMPLE). Investigators can also provide oligos, cDNA clones or cDNA PCR products for custom arrays. PCR products must be received in 384-well Genetix plates (X6004), 20µl at 500ng/µl in 20% DMSO. A completed microarray fabrication requisition and Excel spreadsheet containing the oligo/clone information must be received with the samples. Investigators providing BAC & cDNA clones for custom arrays must complete the microarray fabrication requisition and also provide an Excel spreadsheet containing the clone information. All BAC & cDNA clones must be received in 96-well plates as frozen glycerol stocks and be certified phage free. Please include primers needed for PCR amplification of the inserts and growing requirements of the clones.

Training - The facility will offer training for individuals interested in performing array technologies in their own laboratory. Training will be held monthly for RNA/DNA preparations, probe generation and purification, hybridizations and scanning. Additional sessions will also be held to train users in bioinformatics and microarray data analysis. Individuals will be charged for the reagents required to perform microarray analysis as part of our cost recovery charge back system.

RNA - 1 µg total RNA and a completed sample requisition is required per Agilent expression array. Note: It is required to treat RNA samples with DNase I. All RNA samples must have a bioanalyzer rRNA [28S/18S] ratio > 1.0. It is required that the user provide the concentration and the volume of the sample.

DNA - 1 µg high molecular weight genomic DNA and a completed sample requisition is required per BAC, NimbleGen or Agilent aCGH hybridization. It is required that the user provide the concentration, A260/A280 ratio and the volume of the sample. Low molecular weight DNA samples can be analyzed (i.e. paraffin, formalin fixed tissues), but results may be more difficult to interpret.  The facility cannot guarantee aCGH results from low molecular weight DNA samples.

Samples - A minimum of 10⁶ cells and a completed sample requisition are required for RNA/DNA preparation from cell lines. A minimum of 25mg of tissue is required. All samples must be fresh or snap frozen in liquid nitrogen and stored at -80°C.

Data Analysis - The facility will utilize the in-house clone database or investigator-generated custom clone database along with Imagene and GeneDirector software to locate and identify each spot on an array. After clone assignment, each spot will be normalized. A scatter plot will be generated along with a spreadsheet detailing expression ratios of all normalized spots. This spreadsheet along with supporting image files will be available for data analysis by investigators. The facility's bioinformatics staff cannot be expected to clearly understand the subtleties of each investigators's experiments. Therefore, it is up to each investigator to interact with our bioinformatics staff to complete the final data analysis. Our approach analyzes each experimental parameter to ensure high data quality. Some of the parameters measured are analyzed the same way for many different kinds of experiments. Others, especially time trial studies, depend strongly on the experiments being done and the decisions from the experiments that can only be made by the investigator.

Genomic Services

Requisitions - All high-density screening and BAC or PAC clone requests must be received with a completed, signed requisition. Information for marker sequences to be used for overgo design must have a corresponding spreadsheet in a format consistent with the example described below. Libraries to be screened are selected on the Genomic Screening requisition.

Probes - cDNAs, PCR products, cloned genomic DNA, synthetic oligonucleotides, and end sequence overgos from BACs can be used as probes to screen the genomic libraries. We need about 100 ng of purified probe for labeling (for most probes). The probe should be free of contaminating vector, PCR primers, and other nucleic acids that may create non-specific signals. For oligonucleotide probes, we need about 250 ng for labeling.

Sequence Submission - Genomic or cDNA sequence (preferably 3'UTR) can be used to design overgos for screening the genomic libraries. 95% of markers with sequence >150bp will successfully have an overgo designed. Marker sequence files can be sent as a text, Word or Excel file to the facility in the following format

Clone Requests - Genomic clones can be ordered from the facility by completing the Genomic Clone requisition form. Please select the format you wish to receive the clones (DNA, glycerol, stab).

Clone Characterization - Characterization requests of genomic clones include fingerprinting, PCR verification and sizing of BAC inserts. Completion of the Genomic Clone requisition form is required.

Genotyping Services - Sequenom MassARRAY and Illumina

Requisitions - All genotyping requests must be received with a completed, signed requisition. Information for SNP sequences to be used for oligo design must be emailed in spreadsheet format consistent with the example described below. DNA samples to be genotyped must pass quality control measurements and also have a corresponding spreadsheet provided.

DNA - It is required that the user provide the concentration, A260/A280 ratio and the volume of the sample.  Low molecular weight DNA samples can be analyzed (i.e. paraffin, formalin fixed tissues), but results may be more difficult to interpret and should be discussed before submitting.  Sequenom: 100ng genomic DNA is required for SNP genoyping reaction and 1ug for methylation.    Each microgram of DNA for methylation analysis will be bisulfite treated, resulting in enough tempate for 50 reactions.  Illumina: 250ng genomic DNA is required for targeted GoldenGate SNP genoyping and 750ng for Infinium whole genome genotyping.

SNP Sequence - Simply submit a target SNP sequence(s) for any genome and we will design and manufacture a custom SNP Assay for you. To design custom multiplexes for the MassARRAY and Illumina GoldenGate Systems, you can use SNPs from a variety of sources, including SNPs you already have and those from public databases. 

Methylation Sequence - Simply submit a target meth sequence(s) and we will design and manufacture a methylation assay for you. It is recommended that you provide at least 1,500 bp of sequence and that the region of interest be indicated in the sequence file. Sequemom EpiTyper requires amplicons from 200-600bp, so some CpG regions may have to be assayed using several PCR primers.