Direct Hybridization Assay

Illumina Gene Expression Arrays are designed to be fully compatible with the standard reagents, techniques, and tools that are used extensively throughout the gene expression community, from sample labeling methods to analysis software.

Illumina Gene Expression protocols feature a first- and second-strand reverse transcription step, followed by a single in vitro transcription (IVT) amplification that incorporates biotin-labeled nucleotides. Subsequent steps include array hybridization, washing, blocking, and streptavadin-Cy3 staining.

Direct Hybridization gene expression assays are run on the HiScan or iScan systems.

Fluorescence emission by Cy3 is quantitatively detected for downstream analysis. GenomeStudio software provides results in standard file formats that can be readily processed with most commercial expression-analysis software programs.

Probe design
Candidate probe sequences undergo extensive bioinformatic screening prior to selection. We concatenate 50-mer gene-specific probe sequences with "address" sequences which are immobilized on a bead along with hundreds of thousands of probes of the same sequence.

Direct Hybridization Assay highlights

  • High Reproducibility: r2 value > 0.99
  • High Yield: Multiple hybridizations from a single reaction (> 10 µg from just 50 ng of total RNA)
  • Low Sample Input Requirements: Just 50–100 ng of total RNA required
  • Low Per-Sample Cost: Less than half the price of other commercial arrays

Illumina direct hybridization assay workflow