Kim, Hyun Hee (NCATS)
Chu, Pei-Hsuan (NCATS)
Braisted, John (NCATS)
Kuo, David (NCATS)
This technology includes the use of the Anneal-Pool-Ligate-Sequence method (APLS) to quantify the cellular expression of dozens of genes for high throughput chemical library screening. This method is performed by culturing eucaryotic cells in 384-well format microplates, treating the cells with a library of chemicals, and producing cell lysates. Oligodeoxynucleotide (oligo) pairs representing (21) selected genes, and carrying index sequences for each well (384) and microplate (26), are annealed to mRNAs in cell lysates. Pools of ligated oligo-pairs are amplified by PCR and subjected to “Next-Generation Sequencing” en-masse. Gene sequences are mapped to individual treated cell samples using index sequences for each sample and counted to determine which genes were up- or down-regulated by each treatment.
This technology could be used to identify leads and optimize drug candidates for target selectivity and in vitro safety assessment.
This method demonstrates quantitation of expression using 21 genes in 9984 samples (26 microplates x 384-wells) in a single day using only 26 PCR reactions.