ORB offers highly sensitive and cost-effective gene expression characterization using MI-Ready microarrays for human, mouse, and rat biospecimens. The manufacturing process for these arrays utilizes state of the art oligonucleotide deposition microarray technology and has been optimized to maximize the density and uniformity of oligonucleotide molecules within the circumference of each microarray spot. This deposition technology developed by Microarrays Inc. and combined with ORB's proprietary labeling and array hybridization processing system yields unparalleled sensitivity in detection of low prevalence long RNA.
The Rat MI-Ready array design is based on Operon Biotechnologies' Array Ready Oligo Set version 3.0. This set of oligonucleotides draws entirely from the Ensembl Rat Database Version v19.3b.2 and the Rat Genome Project which is based on the brown rat (Rattus norvegicus) genome.
In contrast, the human and mouse versions of the MI-Ready platform contain whole genome exonic evidence-based oligonucleotide sequences. The oligonucleotide sets were designed by an international collaboration of leading scientists at University of California - San Francisco, Stanford, Stowers Institute, Rockefeller University, and the University of Basel in Switzerland and use a transcriptome-based annotation of exonic structure for genomic loci.
|Highlights of the Technology
Sensitive and Reproducible
Validated by Real-Time qPCR
Summary of Microarray Content
The core content for both Human and Mouse MI-Ready arrays consists of ~30,000 different oligonucleotide sequences (Table I, Probes against constitutive and multiple exons), each designed to detect the RNA product of a unique genomic locus. Additional exon-specific oligo probes are included on the human and mouse microarrays which were designed to detect known alternatively spliced versions of the core transcript set (Table I, Probes against alternatively spliced exons). The probe count for control oligos (the bottom of Table I) generally consist of multiple depositions of several different oligo sequences for each target RNA class. The Rat MI-Ready array is comprised of 34,491 annotated probes designed to detect transcripts from rat mRNAs annotated in ENSEMBL release 37 plus 4,727 unannotated rat-transcriptome specific probes. The core set of 22,167 probes against constitutive exons and 4,795 probes spanning multiple exons were developed by Operon Corp, whereas the "Other" probes were added by Microarrays, Inc.
Table I. Coverage with MI-Ready microarrays for human, mouse, and rat.
|Probe Type||Human MI-Ready||Mouse MI-Ready||Rat MI-Ready|
|Probes against constitutive exons||19,207||24,958||22,167|
|Probes against alternatively spliced exons||8,441||4,201|
|Probes spanning multiple exons||11,511||5,153||4,795|
|Other (BCR/TCR, mtDNA, rRNA, tRNA, ncRNA)||843||766||12,256|
|Transgenes & Vector Sequences||102||37|
|Positive (human or mouse)||864||392|
|Negative (random sequences)||410||97|
Note - the Other category for MI-Ready Rat consists of 7,529 and 4,795 annotated and unannotated probes directed against rat transcripts. For human and mouse microarrays it consists of probes directed against the RNA classes listed in the first column.
MI-Ready 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 the content of the human and mouse arrays visit the website of the lead array designer, Ash Alizadeh of UCSF, and additional specifications about the rat array can be found on the Operon datasheet.
Sample Quality Control
Scientists at ORB 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. Intact RNA is expected 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.
Preparation of Complementary RNA
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). ORB's process, based on the method of Van Gelder1, 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 & Hoffman2, 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.
Microarray Hybridization and Detection of Bound cRNA
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.