For gene expression profiling of mouse and human RNA samples, ORB uses the mouse MEEBO (mouse exonic-evidence based-oligonucleotide) and human HEEBO (human exonic-evidence based-oligonucleotide) 70-mer oligonucleotide- deposition microarrays, respectively. Oligonucleotide sequences for the arrays were designed by an international collaboration of researchers at UCSF, Stanford, Stowers Institute, Rockefeller University, and the University of Basel in Switzerland.

This table shows the number of printed spots on each microarray that contain oligos complementary to different classes of target RNAs. The probe count for control oligos (the bottom of the table) generally consist of multiple depositions of several different oligos sequences for each target RNA class. In contrast, the core content for both MEEBO and HEEBO (the top of the table) consists of ~30,000 different oligonucleotide sequences (Constitutive Exonic, mRNA), each designed to detect the RNA product of a unique genomic locus. Additional exon-specific oligo probes (Alternatively Spliced Exonic) are included on the microarrays, and were designed to detect known alternatively spliced versions of the core transcript set.

The MEEBO & HEEBO microarrays used at ORB are printed on a premium epoxide glass by Microarray, Inc. Each lot of microarrays is quality control tested by Microarray Inc. on a variety of performance measures. Most significantly, 99.5% or more of the array features must be deposited properly on the array. The variety of control oligonucleotide probes on these well-designed arrays enable easy orientation of array images, and the assessment of hybridization stringency, detection sensitivity, and strand specificity. For extensive information on array content please visit the website of the lead array designer, Ash Alizadeh of UCSF.

We check RNA integrity by electrophoresis through 1% Agarose - 2% Formaldehyde gels in 1X MOPS buffer. RNA gels are stained with 1:10,000 SybrGold dye and photographed with an electronic gel documentation system. BioRad Quantity 1 software is used to measure the relative contribution of the 28S and 18S ribosomal RNA bands to the total RNA fluorescence signal. We expect intact RNA to show a 28S/18S ratio of >2.0 with >75% of fluorescence intensity in the ribosomal bands. In order to check for protein contamination, OD 260/ 280 ratios are also determined for each RNA sample. A ratio of 1.7 or higher in 10 mM Tris pH 7.5 is acceptable.

In order to achieve maximal sensitivity, and to minimize sample input requirements, ORB has chosen in vitro transcription based-incorporation of biotinylated UTP for production of amplified and labeled complementary RNA (cRNA). Our process, based on the method of Van Gelder ⁽¹⁾ , is outlined in Figure 1. First, an oligonucleotide containing a 5'-T7-promoter sequence and a 3' T24VN sequence is used to prime reverse transcription catalyzed by Superscript II (Invitrogen). Double-stranded cDNA is prepared from the 1st strand product by the method of Gubler & Hoffman ⁽²⁾ , and purified on a PCR purification column (Qiagen). The double-stranded cDNA is then used as a template for in vitro transcription with T7 RNA polymerase using a high yield transcription kit (Ambion) and including biotin-16-UTP in the reaction mixture. Using our optimized variation of Van Gelder's method we routinely obtain between 25-60 micrograms of biotinylated cRNA from each microgram of total RNA, depending on tissue of origin.

Biotinylated cRNA samples are fragmented, diluted in a formamide-containing hybridization buffer, and loaded on to the surface of microarrays enclosed in custom hybridization chambers. Constant rotation in a Robbins Scientific hybridization oven is utilized to achieve uniform sample dispersion across the array surface by bubble/ gravity mixing. This gravity mixing process allows ORB to minimize inter-sample variation due to hybridization as well as non-specific background, while maximizing signal intensity for low abundance RNAs. After 16-18 hours of hybridization, microarray slides are washed under stringent conditions, stained with Streptavidin-Alexa-647 (Invitrogen), and scanned using an Axon GenePix 4000B scanner.