The ArrayPlate Detection Scheme
The ArrayPlate is hybridized with Programming Linkers, which are DNA oligonucleotides with two distinct sequence domains. One half of the Programming Linker has homology to the anchor sequence attached to the surface of the plate. Each spot (shown in a different color above) has a different linker sequence, allowing the Programming Linker to be specifically targeted to each spot. The gene specific portion of the programming linker has sequence complementary to the protection oligonucleotide generated in the qNPA library step.
The qNPA oligonucleotide library is added to the well and hybridizes to the Programming Linkers. The qNPA oligos for specific genes are captured on the discreet spots (shown in pink, orange, yellow and purple). In a typical qNPA library, differing amounts of oligonucleotides are present in proportion to the differing amounts of starting RNA. In the above diagram, the pink spot represents a highly expressed gene, the yellow spot a non-expressed transcript, and the other spots are moderately expressed transcripts.
A Detection Linker DNA oligonucleotide mixture is added to each well and hybridizes with the qNPA library oligonucleotides (shown in green and blue). The mixture contains oligonucleotides with a complementary sequence to a specific qNPA oligonucletide on one half and a common sequence on the other. The Detection Linker oligonucleotides are added in excess to ensure every bound qNPA library oligo is hybridized to a Detection Linker.
A short oligonucleotide with a sequence complementary to the common sequence in the Detection Linkers is added to the well. This universal Detection Probe is conjugated to the horseradish peroxidase enzyme (HRP).
A substrate which produces luminescence in the presence of HRP is added to each well. The luminescence data is then captured using one of our imagers, and precise, quantitative readings are obtained. The various intensities are a result of the differing localized amounts of HRP present in each spot, and correlate directly to the amount of each RNA present in the original RNA sample put into the qNPA process. The data are ready for analysis.
Click here to view a Technical Animation of the qNPA™ ArrayPlate.
