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The Facility houses a complete Affymetrix GeneChip DNA array system consisting of: a hybridization oven, an automated fluidics station allowing parallel processing of four chips and an Agilent GeneArray scanner. GeneChip probe arrays offer high volume and performance by providing high quality probe sequences using factory-manufactured Affymetrix probe array technology. Assistance is also available on microarray bioinformatics (experimental design, MAS5 analysis, datamining using Spotfire).

QC of RNA samples is performed using the Agilent 2100 Bioanalyzer.

Services offered

One-cycle or Two-cycle labelling	User provides total RNA
	Facility performs target preparation, hybridisation, staining and scanning chips
	Chip image and data files provided in MAS5 format on CD
	Data provided in excel spreadsheet format on request
Hybridisation only	User generates target from total RNA. Fragmented cRNA provided to Facility
	Facility performs hybridisation, staining and scanning chips
	Chip image and data files provided in MAS5 format on CD
	Data provided in excel spreadsheet format on request

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Sample submission

Please discuss your requirements with Kaiman BEFORE submitting samples.

Submit to BRF

• Microarray order form completed and signed
• Samples - RNA should be purified using RNeasy columns (Ambion) prior to submission
• Gel picture to confirm that your RNA is of sufficiently high quality to proceed
• GeneChips

Amount of sample to supply to the BRF
One cycle labelling: Total RNA -5ug at a concentration of 500ng/ul
Two cycle labelling: Total RNA - 150 ng at a concentration of 30ng/ul
Hybridisation: Fragmented cRNA -15 ug (excluding total RNA carry-over).

Upon receipt of samples they will be stored at -70 deg and will be processed in turn. Please ensure that you discuss estimated turn-around times and any other special requirements with Facility staff when submitting your samples.

Test3 chips: Supplied by BRF

GeneChips: We prefer users to supply experimental chips but provided we are given sufficient notice, and the relevant sample submission form and request for chips is received, the BRF can order chips for you. Please contact Karen Edwards (6125 9637, Karen.Edwards@anu.edu.au) prior to sample submission if you require the BRF to order chips.

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GeneChip Expression Analysis Experiments (please visit the WM Keck Facility at Yale for an excellent overview).

Genechip Expression Analysis experiments involve the following major steps:

1) Experimental Design
2) RNA Isolation
3) Target cRNA preparation
4) Hybridisation to the Test array
5) Test array data analysis
6) Hybridisation to the standard array

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Additional information for BRF microarray users

Preparation of your RNA samples for microarray analysis involves a number of steps (RNA purification, cDNA synthesis, IVT and biotin labelling, fragmentation of cRNA, hybridisation, staining and scanning) and takes approximately 2 weeks for one-cycle labelling and 3 weeks for two-cycle labelling from the time we start to process your samples. The time to complete your samples is dependent on work load and instrument status (eg. all four modules on the Fluidics station operational).

The experimental procedure involves approximately 100 different chemicals, enzymes and buffers from more than 7 companies. This means that there are many stages where experimental error or variation may be introduced. For example, total RNA quality and quantity, enzymes, chemicals, column defects, equipment variation, chip batch variation, environmental contamination and operator mishandling.

While all care is taken to ensure that your samples are processed correctly, there is always the chance of unforeseen experimental errors being introduced. QC checks (Bioanalyzer and UV-Vis spectrophotometric analysis) are run at each of 3 steps (total RNA, biotin labelled cRNA and fragmented cRNA). The original source of RNA material can also affect the IVT reaction and it may require more than one attempt to get it to work using different amounts of starting RNA.

You will be updated at 2 stages: 1) after the IVT reaction - proceed with test chip? and 2) after test chip hybridisation - proceed with experimental chip?

Unless there is a problem due to operator error or equipment failure, we cannot take responsibility for samples that do not work and you will be required to pay the full cost for any repeats. If you are not comfortable with this we suggest you prepare samples to the fragmented cRNA stage.

If you have any special requirements please make a note in the ‘Comments’ section of the Service Request Form. Sample prep will be documented and a copy given to you on completion of the experiment.

Useful information

• General information
• Affymetrix GeneChip Expression Analysis Overview
• Protocols
• Data analysis
• Free software for analysis
• Commercial software for analysis
• Spotted cDNA arrays
• Netaffx web site (PIN required for registration – please contact BRF)

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Pricing

Contacts

Experimental and collaborative analyses:
Dr Kaimen Peng
Tel: 0407 212219
email: Kaiman.Peng@anu.edu.au

Bioinformatics:
Dr Stephen Ohms
email: Stephen.Ohms@anu.edu.au