Equipment:
The facility houses a last generation Affymetrix Gene Expression Analysis platform for global RNA expression profiling:
1. GeneChip Scanner 3000 7G with AutoLoader: this instrument performs state-of-the-art density arrays, enabling new applications for genotyping with up to 500,000 SNPs, tiling arrays for transcription, and all-exon arrays for whole-genome analysis. The New GeneChip Scanner 3000 7G incorporates advanced space-saving design improvements, superior speed and performance, improved autofocus, accurate girding and lower scanner-to-scanner variation, to dramatically enhance throughput and efficiency in genetic analysis.
GeneChip Scanner 3000 7G
2. GeneChip Hybridization Oven 640: the Hybridization Oven 640 holds up to eight rotating probe array cartridge carriers (each with eight cartridge slots) for controlled hybridization of up to 64 probe arrays. This unit delivers precise temperature control for consistent performance across all probe array applications.
GeneChip Hybridization Oven 640
3. Dell OptiPlex GX270 computer workstation: is fitted with the Affymetrix Microarray Analysis Suite GCOS software package and internet connection, for measured array scanner data collection and analysis; it also controls the automated steps on the fluidics station and scanner.
4. GeneChip Fluidic Station 450: the Fluidics Station 450 contains four modules. Each module can hold one Gene Chip probe array cartridge and can be controlled independently by the Affymetrix GeneChip Operating Software. The Fluidic Station simplifies the handling of staining and antibody reagents to reduce direct hands-on activities and interruptions for the user.
GeneChip Fluidic Station 450
5. NanoDrop Spectrophotometer ND-1000 : the NanoDrop ND-1000 is a full-spectrum (220-750nm) spectrophotometer that measures 1ul samples with high accuracy and reproducibility. It utilizes a patented sample retention technology that employs surface tension alone to hold the sample in place. This eliminates the need for cumbersome cuvettes. It has the capability to measure highly concentrated samples without dilution.
NanoDrop Spectrophotometer ND-1000
6. Agilent 2100 Bioanalyzer : the Agilent 2100 bioanalyzer is the industry standard for RNA sample QC and has replaced laborintensive gel electrophoresis for this application. It is also rapidly replacing gel electrophoresis for DNA fragment analysis and SDS-PAGE analysis of protein samples. A unique feature of the Agilent 2100 bioanalyzer is that it can be used for both electrophoretic separation and flow cytometric analysis of cell fluorescence parameters. This versatility makes the Agilent 2100 bioanalyzer an indispensable tool for the molecular biologist and biochemist.
Agilent 2100 Bioanalyzer 5
Work flow:
The Core places emphasis on ensuring upfront sound experimental design, as well as expert data analysis and statistical evaluations of the outcome. Thus, initial discussions between users and Core personnel are encouraged to allow for a smooth workflow and effective final data analyses. Intermediate steps of sample preparation, hybridizations, and data acquisitions are performed by the Core personnel.
One cycle of array processing includes:
First day:
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quality and quantity control of all samples
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cDNA synthesis, purification and IVT synthesis
Second day:
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purification, quality control, fragmentation of cRNA, overnight hybridization.
Third day:
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staining, washing;
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scanning;
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absolute analysis.
Three such cycles of 8 arrays each per week will be feasible, bringing the maximum capacity to 24- 32 arrays/week, or approximately over 1000 arrays/year.
Management of Final Data:
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data obtained during quality control and quantification ( Bioanalyzer output)
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raw data obtained after scanning(.DAT, .CEL, .CHP and .RPT generated by the Affymetrix GCOS software)
Data collected will be handed to the Investigator as well as stored at the Facility for backup. Data storage and management issues are coordinated by CBM bioinformatics personnel.
