We use Illumina technology for our microarray services. The Illumina approach for gene expression arrays is as follows:
- An oligonucleotide targets a single gene transcript and is synthesized and conjugated to a micrometer diameter silica bead.
- An oligonucleotide with a unique sequence (molecular barcode) is also conjugated to the same bead.
- Millions of beads for thousands of gene targets are synthesized in parallel and then mixed together in equal proportion.
- Beads are spread onto the slides, enter the holes, and are held by van der Waals forces.
- The beads are positioned randomly, and each gene is targeted by multiple beads.
- In gene expression experiments, a fluorescently labeled RNA transcript is hybridized to the array, and its signal read by an array scanner.
To find the bead to gene correspondence, a clever decoding strategy is used. All beads have a unique barcode, but there are subsequences that are shared between each. One oligonucleotide will connect to the green dye, and the other will connect to the red dye. These oligonucleotides are then hybridized to the array, where their signal is read. Roughly half of the beads will have a green signal, the other half will signal red. The array is then subjected to denaturing conditions to remove the annealed oligonucleotides. The process is then repeated with a different pair of green and red strands. Again, half of the beads will have either a green or red signal, but the signal may not be same as the previous iteration. For example, a bead may have four signal sequences––red-green, green-red, red-red, or green-green––dividing all beads into four groups. Using successive hybridization cycles, millions of beads can be individually decoded to identify which gene transcript they target. The same method applies for decoding beads in genotyping or methylation assays.
Sample Submission Guidelines
Below, are our submission guidelines for those interested in our microarray services.
For DNA samples for genotyping:
- All DNA should be normalized to 75ng/ul, if using the Nanodrop (or 50ng/ul if using fluorescence/picogreen) in TE (10mM Tris, 1mM EDTA) or DNase/RNase-free water.
- The minimum volume should be 15μl.
- If 260/280 ratio is lower than 1.7, the DNA should be purified to remove protein contamination.
- Plate DNA into a 96 well, 0.2ml V bottom, skirted plate (Fisher #AB-0800, USA Scientific #1402-9800, or World Wide Medical Products # 41081006) in columns (A1, B1, C1, D1,…), and label the plate with its specific ID.
- Fill out the sample submission form, including a fund number.
- Seal the plate with an adhesive foil seal, which can withstand being on dry ice or in -80 degrees (Beckman Coulter #BK538619). Make sure the seal is secure by pressing down on each individual well with your finger.
For shipping samples:
- Wrap plates in paper towels or bench pads individually, freeze the sealed plates, -20 degrees, overnight.
- Stack multiple plates with extra padding between the plates so that the wells of one plate do not puncture the seal of another.
- Ship DNA on dry ice. Make sure there is ample dry ice to keep the DNA frozen during the shipment. Make sure to place the DNA plates in a water tight bag.