Microarray technology is a hybridization-based method that can quantitatively measure the expression levels of tens of thousands of genes in a single experiment. Microarrays are constructed on solid substrates, such as silicon or glass, and contain short oligonucleotide sequences that are complementary to every targeted transcript. Each microarray assay requires as little as 50 to 100 nanograms of total RNA and can be performed for a few samples, several hundred samples or thousands of samples.
The Gene Expression Core (GEC) currently supports gene transcript measurement using Affymetrix, Illumina and Agilent microarray platforms. On those platforms, GEC services include 3'-based mRNA, whole-transcriptome and miRNA analyses on total RNA from fresh-frozen and paraffin-embedded tissues.
Illumina gene expression analysis
Direct Hybridization Assay (Direct Hyb). The Illumina Direct Hyb labeling method performs 3'-based gene expression measurements through reverse transcription and in vitro transcription techniques that incorporate biotin-labeled nucleotides into the nascent products.
Labeled complementary RNA (cRNA) products are hybridized onto bead arrays, washed and stained with streptavidin-Cy3. The assay requires 100 to 500 nanograms of high-quality total RNA. Illumina currently makes available whole-transcriptome analysis products for humans, mice and rats.
Whole-Genome cDNA-mediated Annealing, Selection, Extension and Ligation (DASL). The Illumina Whole-Genome DASL assay provides gene expression measurements from highly degraded RNA human samples, such as those derived from formalin-fixed, paraffin-embedded tissues.
Two hundred nanograms of degraded total RNA is reverse transcribed with biotinylated primers. The resulting cDNA product is then annealed to chimeric query oligonucleotides — which contain a gene-specific region and a universal primer sequence for PCR amplification — and bound to streptavidin-conjugated paramagnetic particles. Gene-specific oligonucleotides are extended by second-strand cDNA synthesis and then ligated.
Subsequently, the products are sequestered by magnetic separation and then amplified by PCR with fluorophore-labeled universal primers. The resulting PCR products are purified, hybridized onto Illumina HT-12 v4 BeadArrays and scanned.
Affymetrix whole-transcriptome gene expression analysis
3' IVT Express. The 3' IVT Express labeling kit is used for dozens of Affymetrix microarray products, including the highly popular Human Genome U133 Plus 2.0, Rat Genome 230 2.0 and Mouse Genome 430 2.0.
One hundred nanograms of high-quality total RNA is used for reverse transcription to generate cDNA. Subsequently, the products are in vitro transcribed, resulting in biotin-labeled cRNA. The IVT products are then fragmented and hybridized onto the GeneChips. The arrays are washed and stained with streptavidin-phycoerythrin and then scanned.
MicroRNA (miRNA). One of the methods used by the GEC for microRNA expression measurement is the Affymetrix miRNA Array. This product interrogates the miRNA transcriptome of 71 different species.
The platform requires 100 nanograms to 1 microgram of total RNA for the FlashTag labeling procedure from Genisphere. A polyA tail is added onto RNA fragments and the products are ligated to a Genisphere 3DNA molecule-forming biotin-labeled RNA. The products are then hybridized onto the arrays and subsequently scanned.
Whole-transcriptome labeling. Affymetrix provides two options for whole-transcriptome labeling of human, rat and mouse samples: the Exon 1.0 ST Array and the Gene 1.0 ST Array.
The Exon 1.0 ST Array provides information on both gene expression and alternative splicing, utilizing more than 5 million unique probes and 1.4 million probe sets to interrogate more than 1 million exon clusters.
The Gene 1.0 ST Array utilizes approximately 26 probes that span the entire length of each gene. This array contains a subset of the probes from the Human Exon 1.0 ST Array and includes only the well-annotated content.
Other array types. The GEC can also provide support for gene expression measurement on arrays not previously mentioned. Two Axon 4000B scanners are available for imaging some types of Agilent arrays as well as others that are in a 25-millimeter by 75-millimeter format.
The cost for each project is dependent on experimental design, vendor costs and time required for completion. This information is provided in each project proposal, which must be approved by the principal investigator prior to project initiation.
For more information or to initiate a project, contact us at 507-255-1086 or by email.