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Genomics/Microarray Facility
Services
Contact: Celia Chang, Ph.D.
Tel (215) 898-3902
Fax (215) 898-4521
Chang@wistar.org
The Genomics Facility was established in March of 1999. Dr. Celia Chang, a Ph.D. biochemist, has been working for the facility since 1999 and has the primary responsibility for Illumina gene expression, SNP Genotyping and CNV studies, custom cDNA array production and facility management. Dr. Chang is assisted by Tran Ngyuen, Calen Nichols, Shere Billouin, and Wen-Hwai Horng. Questions related to services should be directed to the facility director, Dr. Chang.
Gene Expression and Genotyping Studies
Illumina Bead Array Platforms
(http://www.illumina.com/pages.ilmn?ID=208)
Hybridization/Imaging Services
Gene expression analysis using microarrays, including every step from mRNA amplification and labeling through hybridization, washing, and image acquisition, is available at the Genomics Facility. Investigators are asked to choose the Illumina Beadchips that best suit their needs and provide information about the quantity and quality of the RNA being submitted. The average time required for hybridizations, starting with sample submission and ending with an image of the array, is 10 working days; however, this time will vary based on the quality and quantity of RNA.
The Sample RNA form should be printed out and accompany all RNA samples submitted for analysis.
Samples submitted for analysis should be provided in eppendorf tubes clearly labeled with the following information:
- species and source of RNA sample;
- purification procedure and whether RNA has been DNAse treated;
- concentration of RNA/DNA.
**The Sample RNA form, and a picture of the gel on which the RNA/DNA was run should accompany the RNA.
Nanodrop and Bioanalyzer analysis can be provided.
Please contact Celia Chang to arrange hybridization of your RNA or DNA samples.
Solexa Genome Analyzer: ( http://www.illumina.com/pages.ilmn?ID=250 )
- ChIP-Seq Analysis
- Paired-end applications
All sample preparations for GAII analysis are provided by the facility. User prepared samples are also accepted. Please email the GAII Submission Form and consult with Calen Nichols nichols@wistar.org for sample requirements, the quality control issues that need to be addressed in sample preparations,and the projected schedule for sample processing. A short description of the project should be provided with the samples.
Capillary DNA sequencing and Microsatellite Analysis on ABI 3130xl Genetic Analyzer
Contact: Ms.Tran Nguyen at DNA_SEQ@wistar.org
Tel (215) 898-3991
Fax (215) 898-4521
The facility uses an ABI 3130xl capillary machine for Sanger-type sequencing with fluorescently-labeled dye-terminators, the ABI four-color detection, and sequence analysis system. The ABI 3130xl is a 16 capillary machine that
can sequence 700-750 clean basepairs for over 100 sequences in a day. The 3100xl can also be configured for fluorescent fragment detection and microsatellite analysis and is equipped with Gene Scanning Software.
How to use the Sequencing Facility
Click here for the DNA Sequencing Submission Form – The completed form must be emailed to DNA_SEQ@wistar.org, and a hardcopy must also be delivered with the samples.
Purified DNA is provided by the investigator with an order form specifying the quantity, form (double-stranded, single-stranded, or PCR product) and estimated size of the DNA. It is essential that the size and concentration of PCR products are determined accurately prior to sequencing. Lambda or Bac clones require submission of greater quantities of DNA. Click here for pricing information.
Procedures
For each sequencing run, investigators must provide in numbered or labeled 1.5 ml Eppendorf tubes:
- double-stranded DNA: 1-1.5 µg of purified DNA at 0.2- 0.25 µg/µl
- single-stranded DNA: 1 µg of purified DNA at 0.1- 0.125 µg/µl
- PCR products: at least 15ng of purified DNA per 100 bp of PCR product in 5ul water.
- Lambda phage or Bac clones: 10ug purified DNA at 2-2.5 µg/µl
A name designation of 8 or fewer characters and the primers to be used for each run must be clearly indicated on the submission form.
- M13 forward, M13 reverse, T7, T3, and SP6 primers are provided by the facility at no additional cost. Specific primers must be supplied by the investigator at 3.2 pmoles/µl.
- Microsatellite Analysis: at least 450ng of DNA at 30ng/ul.
- Depending on well availability, DNA samples delivered by 11:00 am will be available the next day at 3:00 pm. DNA samples from Wistar Cancer Center Investigators only, for which the dye-terminator reactions have been run by the investigator, can be delivered before 2:00 pm for pick up the following day at 2:00 pm.
What to expect from the Sequencing Facility
- For clean double-stranded templates, extremely accurate sequence reads of >=600 bases are obtained routinely.
- Text-files of sequence data are e-mailed.
- The original result folder from the ABI basecaller is placed in a computer folder for electronic retrieval if needed.
- Other modes of delivery can be arranged.
Real-Time PCR and miRNA profiling using ABI TaqMan low density arrays (https://products.appliedbiosystems.com/ab/en/US/adirect/ab?
cmd=catNavigate2&catID=600694 )
and /or SuperArrays (www.superarray.com/) using the ABI 7900HT Fast Real-Time PCR System with 384 well microfluidic PCR card capabilities.
Other Services:
- Protocols:
RNA preparation, hybridization, RNA amplification
- Trouble shooting:
Send an email with your questions to arraysupport@wistar.org
- Training:
By appointment only, hourly cost basis.
- Grant applications:
Core descriptions and facility generated array figures can be provided for grant applications with sufficient notice. Email lshowe@wistar.org with questions.
Array Analysis
Contact: Wen Hwai Horng, M.A., M.S.
Tel: 215-898-3902
Fax: 215-898-4521
Wen-Hwai Horng will carry out basic analysis of arrays, including normalization and simple comparisons on a fee for array basis. He serves as an interface with the Bioinformatics Facility and prepares data for
further analysis. Mr. Horng submits data for publication requirements and is responsible for data transfer to users. The requests for analysis should be discussed when samples are submitted and directed to
Chang@wistar.org.
What to expect from the Facility
The array report forms in Microsoft Excel will be in the format requested by the user.
Special Considerations
Facility Software for Expression and Sequence Analysis
In addition to the array analysis software, the Facility uses several programs for sequence alignment, primer design, and gene annotation. Mr. Horng is also available to train and assist investigators in the use of facility software to carry out their own analyses. Labs are encouraged to take advantage of this
training. Complete pricing information for training and for outside and commercial users is available.
Useful Online Programs
MIA-Molecular Information Agent
The Molecular Information Agent (MIA) searches biological databases to find information regarding a macromolecule. MIA supports search by molecule identification number, gene symbol, sequence, or keyword.
TIGR - The Institute for Genomic Research
This web site offers free (to not-for-profits) gene analysis software such as Glimmer, a gene locator, and TIGR ArrayViewer for visualizing microarray data (requires purchase of MatLab to access).
Statistical analysis of microarray data (SAM)
SMD Microarray Links
Classification of Expression Arrays Version CLEAVER 1.0
BRB Array Tools
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