Each array of this type uses a subset of the probes from a corresponding Exon array to interrogate transcripts from approximately 28,000 well-annotated genes.  Each transcript is probed by approximately 25 probes from exons distributed across the gene.  Transcript level, but not exon level, expression values can be generated from the data.  Mismatch (MM) probes are not present on the array.  A “whole transcript (WT)” protocol (described below) is used to process submitted RNA.  Exon arrays are available for human, mouse and rat profiling. Please see the Pricing page of this site for kind of chips that are available and for their prices.

RNA Isolation - The analysis in the Core begins with total RNA that is provided by an investigator. In our experience, RNA prepared using the Trizol reagent followed by additional purification using RNeasy from Qiagen yields RNA that produces satisfactory microarray data. Modifications of those methods, as well as other methods are also known to produce suitable RNA. Please inquire if you are uncertain as to whether an RNA extraction method that you are considering is suitable.

RNA Amount - 1 microgram of total RNA in a volume of 5 µl is required.

Quality Control - Two quality control measures are carried out on a small aliquot of the submitted RNA samples: (1) a spectrophotometric analysis to confirm the concentration and to detect contaminating proteins and other molecules, and (2) a size fractionation procedure using a microfluidics instrument (Agilent Technologies) to determine whether the RNA is intact. An investigator will be notified if one or more of his/her samples are deemed unacceptable for analysis.

Laboratory Process –

1. Control RNA's - the Core spikes in four in vitro synthesized, polyadenylated B. subtilis mRNA's into each submitted sample.
2. cDNA synthesis I - double-stranded cDNA is synthesized from the RNA sample starting with T7 promoter-tailed random hexamers.
3. cRNA synthesis - cRNA is synthesized in a T7 polymerase catalyzed reaction containing unmodified ribonucleoside triphosphates.
4. cDNA synthesis II - single-stranded cDNA is synthesized from the cRNA template using random primers and dUTP, dTTP, dATP, dCTP and dGTP.
5. cRNA hydrolysis - the cRNA is hydrolyzed by RNase H, and the single-stranded cDNA is purified.
6. Fragmentation of a single-stranded cDNA - Uracil DNA glycosylase and Apurine/Apyrimidinic Endonuclease I are used to fragment the cDNA at Uracil-containing positions.
7. Biotinylation - The fragmented cDNA is end-labeled with a biotinylated nucleotide analog.
8. The fragmented cDNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
9. The chips are then transferred to a fluidics instrument that performs washes to remove cDNA that has not hybridized to its complementary oligonucleotide probe. The bound cDNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
10. Each cDNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a file-system from which it can be downloaded via a password-protected URL.