The University of Toledo : Genomics Core Lab

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Genomics Core Lab - Sample Submissions

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	Genomics

We suggest that you contact us directly, if you would like to design a custom microarray project. Please contact the GCL Director by e-mail, David.Weaver@utoledo.edu, or by phone: David Weaver, D.D.S., Ph.D., 419-383-6105.

What you need to provide for the Affymetrix System

We recommend that you provide biotin-labeled fragmented cRNA in a quantity sufficient to allow for one time application of the hybridization cocktail to each chip. For example, a single human sample to be run on the GeneChip® Human Genome U133 Plus 2.0 Array would require 20 µg of biotin-labeled fragmented cRNA (final adjusted cRNA by the Affymetrix protocol). In this example, 5 µg would be used to run a Test3 chip (required by the GCL) to assess sample quality before applying 15 µg to the standard genome chip. Specific requirements for paperwork accompanying the sample are listed below.

Minimally, we can accept 15 µg of prepared cRNA for creating a hybridization cocktail that will be reused on the several chips (above example Test3 and U133 Plus 2.0 arrays.) 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:

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 need between 1-15 µg of good quality total RNA for each sample for One-Cycle Target Labeling or 10 - 100 ng total RNA for Two-Cycle Target Labeling. This enables the researcher to obtain a sufficient quantity of labeled cRNA for target assessment and hybridization to arrays.
Spectrophotometric analysis at 260 nm and 280 nm of the cRNA (In Vitro Transcription or the 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). For quantification of cRNA when starting with total RNA, a required adjusted cRNA yield must be calculated to reflect carryover of unlabeled total RNA. This is outlined in the Affymetrix protocols and the researcher can contact the GCL for help if required. This adjusted cRNA yield is extremely important for determining the amount of cRNA to be utilized in the subsequent fragmentation step.
Calculations concerning fragmentation of cRNA. Using the adjusted cRNA concentration, the suggested final concentration of RNA in the fragmentation mix should be 0.5 µg/µl. Remaining calculations determine the final fragmented cRNA concentrations used for the hybridization cocktail.
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 cRNA (IVT product).
C. Fragmented cRNA.
D. Appropriate size RNA markers to determine size of fragmented cRNA .

What the GCL provides:

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.
Biotin Labeled cRNA and Test3 array
Samples that pass the QC and Quantitation requirements will be required to first be hybridized to an Affymetrix Test 3 array to check for transcript integrity. Transcript quality is assessed by comparing the 3'/5' ratios of a collection of housekeeping genes. This step is required to avoid costly application of an unsuitable sample to the more expensive standard arrays. Again, clients will be notified immediately if the transcript quality in unsuitable for the 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. Comprehensive lists of genes that are significantly regulated in TEST vs. REFERENCE samples will be generated per the researcher's specific requests.

Page updated: November 03, 2008

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Program in Bioinformatics & Proteomics/Genomics : Genomics Core Lab - Sample Submissions