This assay relies on the extensively well tested and validated Illumina cDNA-mediated Annealing, Selection, Extension, and Ligation (DASL) protocol. Custom panels are easily designed for profiling 32–384 transcripts at a time. The DASL Assay is robust to partially degraded RNA and provides equally reliable results from valuable FFPE samples.

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How Does This Work?

The Basics

The DASL assay is nearly identical to the standard Golden Gate genotyping assay, but includes an additional first step whereby the mRNA is reverse-transcribed, using biotinylated random primers, into activated cDNA.

At this point, the Illumina Golden Gate assay combines the activated cDNA with allele-specific oligo primers targeted to your regions/loci of interest. These oligos are used to amplify and fluorescently label each targeted locus. This step can incorporate up to 384 loci in one reaction well for each DNA sample, a process called “multiplexing”.

Then, these amplified alleles are hybridized to and immobilized on a special substrate built by Illumina, called a VeraCode bead. From here, the Illumina BeadXpress machine can automatically determine the identity of oligo primer hybridized to the VeraCode beads, and measure and quantify the fluorescent signal (thus indicating level of expression) for each amplified locus. In general, the assay can distinguish between low, medium and high expression levels, independently for each locus. Further quantification of expression can sometimes be achieved with additional bioinformatic analyses.

For a more detailed description of the mechanics and chemistry, we recommend that you consult the Illumina web site.

How to Get Started

Our clients have a choice in this. Either, they develop their target loci list and flanking oligos independently, or they choose to have our bioinformats specialists assist them in this process.

Once an oligo list has been created for the loci of interest, this file is provided to Illumina as one part of the order. Illumina will use the SNP and oligo list to create an DAP (DASL assay pool) which is actually the list of which VeraCode beads will match to which locus oligos.

Illumina then sends the DAP and associated reagents to our laboratory. Once you’ve sent your RNA, we perform the cDNA synthesis and bisulfide conversion steps and initiate the Golden Gate assay using your DAP. When your data is generated and analyzed for accuracy, we send it to you.