Genomics Core Lab - Sample Submissions
ANALYSIS OF MICROARRAY DATA
We suggest that you contact us directly, if you would like to design a custom microarray
project. Please contact the GCL Director by email, firstname.lastname@example.org, or by phone: 419-383-6105.
What you need to provide for the Affymetrix System
We recommend that you provide biotin-labeled fragmented aRNA in a quantity sufficient
to allow for one time application of the hybridization cocktail to each microarray
cartridge. For example, a single human sample to be run on the GeneChip® Human Genome
U133 Plus 2.0 Array would require 12.5 µg of biotin-labeled aRNA after completion
of the Affymetrix protocol. Specific requirements for paperwork accompanying the sample
are listed below.
Minimally, we can accept 12.5 µg of Affymetrix kit-prepared aRNA for creating a hybridization
cocktail that, if needed, can be reused on several chips. Affymetrix has informed
us that a hybridization cocktail may be used up to 5 times without significant loss
of signal for transcripts of average abundance.
Required information with submitted sample:
Information may be obtained by the following method, or can be substituted with appropriate
information from instrumentation, such as the Agilent Technologies 2100 Bioanalyzer.
What the GCL provides:
- Spectrophotometric analysis at 260 nm and 280 nm of the isolated total RNA to determine
sample concentration and purity. (For the extraction of total RNA, we recommend Invitrogen
Life Technologies TRIzol followed by cleanup using a Qiagen RNeasy kit.) The A260/A280
ratio should be close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable). The
researcher will prepare RNA samples with the Affymetrix 3' IVT Express Kit to obtain a sufficient quantity of labeled aRNA for target assessment and hybridization
to arrays. As little as 50ng total RNA is required for amplification. Recommended
starting amount is 100ng total RNA.
- Spectrophotometric analysis at 260 nm and 280 nm of the aRNA (In Vitro Transcription (IVT)
product) to determine sample concentration and purity. The A260/A280 ratio should
be close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable). Sample quantization
is required for determining the amount of aRNA to be utilized in the subsequent fragmentation
- Calculations concerning fragmentation of aRNA. Using the aRNA concentration, the aRNA
fragmentation mix should be created according to the Affymetrix protocol. The final
fragmented aRNA concentrations used for the hybridization cocktails will need to be
completed, and will be recalculated by the GCL.
- Picture of agarose gel stained with ethidium bromide (or equivalent), with lanes containing:
A. Total RNA of starting sample. Should exhibit a smear representing the various RNAs
with both 18S and 28S ribosomal RNA bands clearly visible.
B. Purified aRNA (IVT product).
C. Fragmented aRNA.
D. Appropriate size RNA markers to determine size of fragmented aRNA .
- RNA Quality Check and Quantitation
All submitted samples along with required information will be evaluated by the GCL
Director. Investigators will be notified immediately if the RNA is degraded or otherwise
unsuitable for GeneChip application.
- GeneChip Microarray Hybridization and Data Analysis
Samples that pass the above QC steps will be hybridized to the requested oligonucleotide
chips. Microarray data will be supplied to investigators as Affymetrix unfiltered
complete data sets in a CD format or through an online secure file transfer protocol.
Comprehensive lists of genes that are significantly regulated in experimental vs. control
samples can be generated per the researcher's specific requests.