home services platforms affymetrix: overview & faq
As an Authorised Affymetrix Service Provider, the Microarray Centre brings you the most powerful opportunities to
enable whole-genome discovery at multiple levels to provide answers to your critical research questions. We have
solutions for RNA that are not only limited in quantity but also RNA which are somewhat degraded to severely
degraded in quality. Prior to submitting your samples we provide free consultation, if needed, to help design
your experiment. Pilot studies are offered that will help you investigate your research for future directions.
Services at a glance:
- Expression at Gene and Exon level
- miRNA profiling
- Genome-wide SNP arrays
- Tiling arrays
- GeneChip® scanning service and Sample Processing Service
This document includes:
- Sample submission guidelines
- Warranties and conditions
- Customer information forms
Please note that this pricing is for academic groups. Commercial customers should contact us for a quote.
Affymetrix Service Coordinator:
Please note that special paperwork is required for shipping RNA from locations outside of Canada. For the Customs documentation that you will need, please download this form . Once you send us this information, the UHN Customs Officer will generate the appropriate Customs document and send it to you via e-mail. This document must be included with the waybill on your package.
For gene expression profiling using Affymetrix GeneChips, we require 20 ng of total RNA for the small scale labelling method (NuGen) and 100 ng of total RNA for the 3' IVT Express labelling method. For ChIP-Chip experiments, we require 9 µg of amplified DNA. For GWAS assays (SNP arrays), 500 ng of total genomic DNA is required at a concentration of at least 50 ng/µL. Please inquire about the amount of starting material required for miRNA profiling.
The UHNMAC has a stock of "commonly used" Affymetrix chips that are available only for local users to purchase. Non-local (outside of Ontario) users must purchase the chips directly from Affymetrix but have them delivered to the UHNMAC.
More information about the content of Affymetrix GeneChips can be found on the Affymetrix website at the NetAffx Analysis Center.
GCOS stands for Gene Chip Operating System. It controls instrumentation and allows creating "chp" and "rpt" files.
GCOS offers 2 methods for scaling and normalisation. Global Scaling and Selected Probe Set Scaling. Affymetrix recommends global approach since it is simple and fast. Selected probe set scaling is tedious and complicated in a sense that one always had to make sure same set of probes are selected and often housekeeping genes varies from day to day and from sample to sample.
The 20x Eukaryotic Hybridization Controls are spiked into the hybridisation cocktail, independent of RNA sample preparation, and are thus used to evaluate sample hybridisation efficiency on eukaryotic gene expression arrays. BioB is at the level of assay sensitivity (1:100,000 complexity ratio) and should be called "Present" at least 70% of the time. BioC, bioD, and cre should always be called "Present" with increasing signal values, reflecting their relative concentrations.
Poly A controls are spiked in the reaction to evaluate the RNA quality. They contain both T3 and T7 promoter sequences. When reactions are spiked all should be present except LYS is present only 70% of the time (1:100,000 dilution). Poly A controls and it’s dilutions are as follows:
- DAP is most concentrated 1:7,500
- THR dilution is 1:25,000
- PHE dilution is 1:50,000
- LYS dilution is 1:100,000
Small scale labelling method (either NuGen's Ovation Amp V2 or Ovation WT Pico system) allows you to start with as little as 500pg of total RNA and is beneficial when RNA is limiting. The 3' IVT Express labelling method is a quick and simple single cycle labelling method from Affymetrix that requires 100 ng of total RNA.
Yes, the same quality of performance from the 3' IVT Express Kit can be expected as from the One-Cycle Target Labelling and Control Reagents.
Yes, the QC metrics will be similar. The B-actin and GAPDH ratios may be slightly elevated with the new 3' IVT kit, but still within the acceptable range. (In particular, the B-actin 3'/5' ratio may be higher at lower input amounts.)
Yes, degraded RNA can be processed using a Pico kit from NuGen. For RNA that is somewhat degraded the amounts required are 5-8ng with a RIN of 6 and above. Severely degraded RNA can also be analyzed using this kit however much higher amount of RNA is required. 50ng of total RNA is required in this situation at RIN of 0-4.
We can run 48 samples per week using the 96-sample protocol.
The same assay is used (and samples run on the 5.0 can be re-hybridised on the 6.0).
DNA is digested with NspI and StyI restriction enzymes and ligated to adaptors that recognise the cohesive four base pair overhangs. All fragments from the digest are substrates for adaptor ligation. A generic primer that recognises the adaptor sequence is used to amplify adaptor-ligated DNA fragments. PCR conditions have been optimized to preferentially amplify fragments that range in size from 200 to 1100 base pairs. PCR amplification products for each restriction enzyme digest are combined and purified using activated beads. The amplified DNA is then fragmented, labelled, and hybridised to a Genome-Wide Human SNP 6.0 Array.
A minimum of 44 samples should be clustered and analysed using the Birdseed v1 algorithm. A minimum of 15 female samples should be included for robust gender determination results.
There are 5 different housekeeping genes on every GeneChip array. We are mostly interested in GAPDH and b-Actin. b-Actin (ACO7) and GAPDH are used to assess RNA sample and assay quality. Both should result with signal (3’/5’) ratio close to 1 however, up to 3 is good too. Ratio of 10 is extremely high, indicating sample degradation or RT didn’t run very well.
Note: For 1-cycle (1 µg - 10µg of total RNA) Sig (3’/5”) ratio range from 3-1; For 2 cycle (10 ng – 100 ng of total RNA) Sig (3’/5’) ratio range from 10-8; b-Actin usually results in higher ratio over GAPDH.
Each scanner has a unique inherent electrical noise associated with its operation. Since a significant portion of noise (Raw Q) is electrical noise, values among scanners will vary. Array data (especially those of replicates) acquired from the same scanner should ideally have comparable noise (RAWQ) values. Typically, noise (RAWQ) values range from 1 to 4. However, there are no guidelines.
.dat file: pixilated image and is a raw file (each at 150MB)
.cab file: file that incorporates dat, cel and chp files using DTT (data transfer tool)
.cel file: file where intensities are averaged
.chp file: .cel file analysed with GCOS using global scaling and normalisation method
.rpt file: report file where the QC matrix resides
One can use "cel" and "chp" files to analyse data in GeneSpring.
To analyse the data in GeneSpring, one can use "cel" and "chp" files.
The TGT value is the set target intensity for the scaling and global normalisation of data. There are no set guidelines for choosing a TGT value.; A TGT value between 100-500 (default is 500) can be used and should be kept same among one study. You may want to aim towards a lower scaling factor and monitor this for the entire study.
Scaling Factor will depend on the Target Intensity you set for your global scaling. Default target intensity is set at 500, which gives a scaling factor between 3 and 4. Target intensity set at 150 results in scaling factor of 1 and 2. What is important with the scaling factor is not its actual value but that there is "consistency" among chips. If you regularly get scaling factor of a certain value and then for one chip you see a significant change, it could indicate a problem with that experiment/chip. If the scaling factor is lower then that chip should be "brighter" which could be caused by higher background, if the scaling factor is higher, the chip should be "dimmer" which could be caused by lower hybridisation.
Yes, if you are shipping RNA to Canada you are required to include specific Customs documentation with your shipment. To prepare this document, we require the following information from you: full name, mailing address with postal code, email address, phone number, name of courier (ie. FedEx), the number of packages being sent and the approximate weight and size, and a description of what you are sending (non-hazardous, non-toxic, non-infections RNA or DNA only). Please also specify the organism from which the samples are from and the number of tubes (with the approximate volume of each tube). Please indicate the samples as having zero value. Once you send this information, the Customs Officer at the UHN will generate the appropriate Customs document and send it to you via email. This document must be included with the waybill on your package. If you are shipping from within Canada, customs documentation is not required.