Equipment & Protocols
Illumina BeadReader Scanner
The Illumina BeadReader scanner is designed to read both slide-based arrays and the multi-well Sentrix Array Matrices. It has <1 micron resolution and can simultaneously scan at two wavelengths. Excitation wavelengths are 532 nm and 635 nm. The emission spectrum is 550-600 nm and 650-700 nm. Files are output in 16-bit TIFF images. The limits of detection are 2 fluor/sq. micron. The IMC scanner has been modified by Illumina to allow scanning of high density arrays. Although not relevant to operation, the BeadReader weighs 121 lbs.
Expression Array Protocol
Total RNA is submitted by the investigator to the Illumina Microarray Core (IMC). The concentration of the RNA is verified by the IMC using a NanoDrop spectrophotometer. This step also looks for the appropriate 260 / 280 ratio for RNA and looks for organic and salt carryover in the sample using the 260 / 230 ratio. An aliquot of each sample is run on an Bioanalyzer (Agilent) to observe 18S and 28S ribosomal RNA peaks and to look for degradation of the RNA.
Total RNA that has passed the QC tests will be amplified using the TotalPrep RNA Amplification Kit (Ambion), which can amplify RNA across a range from 50 to 500 ng. The resultant amplified RNA (aRNA) incorporates a biotinylated UTP nucleotide. An aliquot of aRNA is hybridized to an Illumina BeadChip using proprietary buffers, first at 65⁰C for thirty minutes, then overnight at 58⁰C.
Following hybridization, the arrays are washed and then stained with Cy3-streptavidin, washed again, and then scanned on an Illumina BeadStation bead array scanner. Data from the scanner is consolated using the GenomeStudio software. Built in quality control measures are monitored in GenomeStudio to look for obvious problems in the hybridization process. Final output is customized to allow use of a variety of statistical packages, including BioConductor and GeneSifter.
Genotyping Array Protocols
Whole Genome Genotyping
Protocols vary depending upon the array being run, so it is wise to check with the IMC to see how the following applies to a specific product.
DNA is prepared by standard high quality protocols. 200 to 750 ng is needed for each sample, depending upon the size of the array (1). The DNA is denatured and then neutralized for amplification. The denatured DNA is isothermally amplified several thousandfold using a proprietary Illumina protocol. The amplified product is fragmented by a controlled enzymatic process that does not require gel electrophoresis. Overfragmentation is avoided by using end-point fragmentation. Fragmented DNA is recoved by isopropanol precipitation. The fragmented DNA is then hybridized to the 50-mer probes of the BeadChip.
Unhybridized and non-specific DNA is washed from the array. Single base extension of the oligos on the BeadChip incorporates modified nucleotides at the site of the single nucleotide polymorphism. The labeled nucleotides include biotin-labeled ddCTP and ddGTP and 2,4-dinitrophenol (DNP) -labeled ddATP and ddUTP. Following additional washing to remove unincorporated nucleotide, the arrays are stained with Alexa555 and Alexa 647 using a dual color, orthogonal, multi-layer immunohistochemical sandwich assay (2). Stained arrays are scanned using the Illumina BeadReader microarray scanner. The data is consolidated using Illumina's GenomeStudio software.
1. Steemers et al. 2006. Nature Methods 3, 31.
2. Pinkel et al. 1986. Proc Natl Acad Sci USA 83, 2934.