Illumina arrays are based on the ordered arraying of oligo-coated beads. In the DASL assay each oligonucleotide (bead type) represents a a specific gene. Since each bead type is present an average of 30 times on each array, each DASL gene expression level measurement call is thus the result of the mean intensity of all 30 replicates, resulting in industry-leading accuracy, reproducibility, and call rates (all >99 %).
Illumina's c DNA-mediated Annealing, Selection extension and Ligation (DASL) assay is part of a powerful gene expression solution designed to generate reproducible RNA profiles from degraded samples such as formalin-fixed, parafin embedded (FFPE) tissues. Importantly, since the DASL assay works with degraded RNA samples Illumina recommends performing a prequalification qPCR assay to verify that all RNA samples have performed equal in the first strand cDNA reaction that starts the protocol (the genomics Core will perform this qPCR step). The two links provided below provide both a description of the DASL assay and a description of the differences between Illumina's two assays for studying gene expression: the DASL assay and the Whole Genome Gene Expression assay.
DASL-assay description
Sentrix 96 Array Matrix
Illumina offers two different array platforms for the DASL assay: the Sentrix® Array Matrix (to process 96 RNA samples in a single experiment) and the Sentrix® BeadChip (to process 16 samples in a single experiment).
Sentrix BeadChipS
At present Illumina offers one standard DASL assay oligonucleotide panel, the human cancer panel which can measure the expression levels of 502 known human cancer genes. In addition to this cancer panel customers can design their own unique DASL panel of between 512 and 1536 genes to use on one of two Sentrix® Universal Array platforms (the Genomics Core can mediate the design of a custom panel if necessary).
The DASL assay is designed to monitor gene expression in partially degraded RNAs, such as those derived from formalin-fixed, paraffin-embedded (FFPE) tissues. Illumina recommends the High Pure RNA Paraffin Kit from Roche Molecular Biochemicals (catalog # 3 270 289) for these type of samples.
For protocols to isolate intact RNA, please see the Illumina Whole Genome gene expression page on our website.
A) Full Service:
Clients will:
- Perform the RNA isolation and submit their RNA samples (normalized to 40-200 ng/uL with DEPC-treated H2O) in a 96-well plate. Provide at least 20uL of normalized RNA sample.
Genomics Core personnel will:
- Perform the DASL Assay protocol for the RNA samples. This includes prequalifying the RNA samples using qPCR as described under section 2.
- Perform BeadChip/Array Matrix hybridization and washing.
- Perform BeadChip/Array Matrix scan and evaluate the quality of the experiment and produce output files.
Clients can:
- Perform additional data analysis using Illumina's BeadStudio software (available on a Core computer) or analyze the data using other means available to them.
Sample Submission:
Purchase of Illumina BeadChip/Array Matrix & Associated Reagents:
Clients are responsible for purchasing the Illumina BeadChip/Array Matrix and associated reagents. The company contact info is: llumina East, 50 Barnes Park North Wallingford, CT 06492, USA Phone: 203-678-2400 Fax: 203-265-9109 Email: info@illumina.com
We ask that you have the BeadChip/Array Matrix and associated reagents delivered to either Dr. Pieter Faber or Jeanna Guenther-Johnson (NE5-253). After receiving the BeadChip/Array Matrix and associated reagents from Illumina we will inspect the items and properly store them until use.
Every client must fill out an Illumina DASL Assay Request Form and bring this to the Core when submitting RNA sample(s). Per sample 20µl of the RNA is needed which must be provided at a concentration of 40 - 200 ng/µL. RNA samples, accompanied with the request form, should be delivered in person to either Dr. Pieter Faber or Jeanna Guenther-Johnson at NE5-253 where they will be stored at -80°C. Projects will be handled in the order received. At the time of submission a time-frame for finishing the project will be given which will depend on the size of the project and other ongoing Illumina projects.
Data Output Files:
The full service data output file will contain: (i) all data related to the quality control and quantitation of the total RNA and amplified RNA samples that were used in the project, (ii) a summary of the internal control features that are part of each Illumina based gene expression experiment and (iii) the raw image data files generated after scanning the BeadChips. [Other data output formats are possible and can be discussed with the Core manager ( faberp@ccf.org )].
Illumina BeadChip experiments come with built in controls that include negative controls, an extension gap control, first hybridization controls, second hybridization controls, and contamination detection controls. These control oligos (with the exception of negative controls and second hybridization controls) are designed to detect different target sites of the human QARS (glutaminyl-tRNA-synthetase) gene.