The University of Toledo

Genomics Core Lab - Sample Submissions

GCL INTRODUCTION

GCLSERVICES

GCL FORMS

GCL PRICING

GCL INSTRUMENTATION

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, david.weaver@utoledo.edu, 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.

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 step. 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 .

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. 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.