TCGA offers whole Microarray service from Custom design of microarray & support in design arrays, preparation of probe synthesis (cDNA, PCR products and Oligoneucleotide) & quality control, Fabrication of human arrays (standard formats available, custom arrays on demand), target amplification, quality control and labelling (direct and indirect) of target, hybridization, scanning, detection and preliminary data analysis upto advanced data analysis.
Our goal is to offer all aspects of spotted microarray technology from array design and manufacturing to acquisition of high quality data within a minimal time frame. Our state of the art equipment and qualified staff will ensure that your arrays are designed to best suit your project. All aspects of protocols from printing to labeling and hybridization will be optimized to produce high quality results. The facility can support researchers who are making microarrays on 25x75mm glass substrates, using either PCR product from cDNA libraries or Oligonucleotide as probes.
TCGA offers world class service at affordable price to the scientific community to promote research and development and to help novel discovery.
 

There are four main entry points into the microarray core. The first entry is for those who would like assistance with all aspects of array design and construction. The second is for researchers who have determined the optimal design for their array and wish to use the facility for array construction and hybridization. Entry three is for those who posses an array, and would like to begin doing hybridization experiments. The final entry point is for those who would like to acquire data from an array that has already been hybridized.

Experimental Design
Consultation (STRONGLY RECOMMENDED) This stage is targeted to those designing cDNA arrays for use with dual-color detection. The facility staff is available to consult on design aspects of the array such as control genes for normalization of finished data, reagent selection, cDNA library construction, and PCR amplification of cDNA clones. The user will be responsible for the PCR amplification of the cDNA inserts from plasmid or glycerol stocks. The GATC automated cleanup service is available for microarray users who need to purify their printing material

Printing of Glass Microarray Slides
Array construction For those who possess PCR product or oligonucleotides that are ready to be printed onto a microarray slide.  TCGA will fabricate the arrays. The printing material will be lyophilized, and then resuspended in the appropriate printing buffer. Our Bio-Rad Chipwriter Pro can fabricate up to 126 arrays at a time, using as many as 48 Telechem Stealth pins, achieving densities of 30,000 spots per slide. The Chipwriter allows for flexibility in chip layout, with highly reproducible spot morphologies.

Labeling and Hybridization
For those who have already fabricated their arrays, and would like TCGA to perform the hybridizations. Our expert staff will perform the labeling reactions  and direct/indirect incorporation of Cy-3 and Cy-5 dyes. Hybridizations are performed either manually or by Automated Slide Processor (ASP) that allows for the most consistent results possible.

Detection and Preliminary data analysis
Data acquisition can be performed by the facility staff if the user has an array that has already been hybridized. We use the AXON Instruments Scanner 4200 Autoloader. Feature intensity values are extracted from the resulting image using Gene PixPro

Advanced Data analysis and Report Generation
Following the Detection and Quantification of the array image, Advanced Data analysis and Report Generation will be done by using the Hi End software packages by Experienced Scientists in the Field

Technology

TCGA Microarray Facility is equipped with latest technology to meet the standards of the present scientific community.

ESI Arrayer for Printing High Dense Arrays

Contact printing using Stealth pin technology
Temperature and humidity control ensure uniformity of spots
Spots small as 100 microns in diameter
Capacity of 20,000 spots per slide
136 slides per batch
Bar code tracking of plate ensures minimum handling of samples
Choice of printing Duplicate spot to check the reproducibility of the experiment

Axon Microarray Scanner 4200A

Choice of con focal and non-con focal laser scanner and multiple image analysis software.
Compatible with gel and polymer coated, glass and plastic arrays of standard dimensions (1"x3", 25 mm x 75 mm, 0.9 mm-1.2 mm thick).
Image output compatible with all standard data analysis software.

 Data analysis and Report Generation

Data normalization and analysis using high end software packages Advanced Data Analysis and Report Generation Consultant on Array Designing and

Sample Preparation Guidelines

• Samples should be concentrated to 20µg in approximately 10-20µL RNase free water. DO NOT SPEED-VAC RNA! Precipitate RNA using 1/2 volume of 7.5M ammonium acetate and 3 volumes of very cold 100% ethanol (-20deg). RNA does not precipitate well at room temperature, so a 30 minute incubation at -20C is necessary. Centrifuge at maximum g force in a microfuge at room temperature for 20min. Remove the supernatant, and overlay the pellet gently with 250ul ice cold 70% ethanol. Centrifuge at maximum g force for 2 minutes. Remove the supernatant, and dry the pellet for 15-20 minutes at room temperature. Be sure that the pellet is completely dry, but do not over-dry (i.e. speed vac or heat). Resuspend in the appropriate amount of RNAse free water.

• RNA can be stored at -80C for up to one year, but freshly isolated samples are always best. Freeze thawing of RNA can be damaging and should be done as little as possible.

• Another common method used to isolate RNA from difficult tissue is with the use of phenol based reagents like TRIzol or RNAzol. These methods give high yields and purity, but do not remove tRNA and 5s RNA. This can cause inaccuracy in RNA quantitation. Also, residual phenolic compounds can damage reverse transcriptase enzyme, inhibiting the labeling reaction.

• RNA can be quantified by U.V. spectrophotometer or through the use of Ribogreen dye (Molecular Probes) and a fluorometer. RNA should also be run on an agarose denaturing gel to verify the integrity of the RNA.

• Purity can be estimated by O.D.260/O.D.280 ratio. A ratio of 1.8 to 2.0 is typical for pure RNA. Buffer pH can affect this ratio, so it is recommended that readings are taken in 10mM Tris, pH 7.2-7.5

• Design your experiment. You need an experimental sample (treated, diseased, etc.) and a control sample, to which you will be comparing the experimental samples. These control and experimental samples should be as similar as possible, except for the treatment. We advise a minimum of 3 biological replicates and 2 arrays per biological replicate (dye swapped). Optimal replicates would be 5 or more per experimental condition. If you are concerned about replicates contact us. Also confer with core personnel for suggestions on optimum labeling technology. See Suggestions on Sample preparation page.

• Review all suggested protocols and purchase necessary reagents, kits and supplies.

• Isolate your experimental and control cells or tissues and perform RNA isolation as per protocol. For any technical service questions regarding RNA isolation you may contact us.

• Send samples of your RNA to the core facility for analysis of integrity and quantity. See Instructions for RNA Submission.

• Synthesize fluorescent labeled cDNA from your control and experimental RNA samples. You should confer with core personnel for recommendations on optimum labeling technology for your experimental application. Most commercial labeling kits include all reagents and instructions required for producing labeled cDNA from your RNA samples.

• Hybridize your labeled control and experimental cDNAs to the microarray as per protocol, according to the labeling technology used. Most commercial labeling kits include reagents and protocols required for microarray hybridization.

• Immediately following overnight hybridization, perform the stringency and detection washes as per protocol, according to your labeling technology. Most labeling kits include protocols and reagents required for post-hybridization processing.

• At this point in the microarray process the fluorescent dyes hybridized to the arrays are extremely vulnerable to environmental conditions with cyanine-5 being more susceptible than cyanine-3. Carefully handle Processed Arrays for storage procedures that will protect the fluorescent signals on your arrays until they are scanned.

• Send processed array to core laboratory for scanning, data acquisition, and analysis.

• You should receive your data report within 15-20 working days. If you need your results sooner contact us for Rush Service.