Assays

Broad LINCS Center

Large-scale gene expression profiling on the L1000 platform:  The L1000 platform pairs ligation-mediated amplification (LMA) with a Luminex-bead based detection system to allow the quantitation of 1000 mRNA transcripts per well. The L1000 assay is an extension of a previously reported method for expression profiling based on Luminex bead technology (Peck et al., 2006) to create a 1,000-plex profiling solution. 

Briefly, the method involves LMA, using locus-specific probes engineered to contain unique molecular barcodes, universal biotinylated primers, and 5.6-micron optically-addressed polystyrene microspheres coupled to capture probes complementary to the barcode sequences.  After hybridizing the LMA products to a mixture of beads and staining with streptavidin-phycoerythrin, the hybridization events are detected using a two-laser flow cytometer, whereby one laser detects the bead color (denoting transcript identity), and the other laser detects the phycoerythrin channel (denoting transcript abundance).  The plex limit for this approach is set by the number of available bead colors, and was previously 100.  However, in anticipation of the need for higher complexity, Luminex has collaborated with the Broad LINCS Center to produce 500 distinct bead colors and an instrument capable of discerning them.  The Broad LINCS team has tested these reagents and instruments in the laboratory, and find the system to have excellent performance characteristics.  The Broad LINCS Center has also developed a computational strategy that allows for two transcripts to be detected using a single bead color (with subsequent computational deconvolution).  The resulting assay is a 1,000-plex assay detectable in a single well of a 384-well plate at very modest cost.

Harvard Medical School (HMS) LINCS Center

A distinguishing feature of the HMS LINCS Center is its use of a wide range of measurement technologies ranging from direct assays of drug-kinase interaction in cell extracts, to multiplex biochemical assays of cell signaling proteins, to imaging assays, to assays of transcriptional response (in collaboration with the Broad LINCS Center) and cell viability. More information about HMS LINCS assays can be found on the HMS LINCS website. Note that it is not possible to collect all types of assay data for all perturbations and cell types. Instead we follow an adaptive data collection strategy in which exploratory studies are used to focus subsequent experiments on areas of cell-perturbation-measurement in space in which we believe that informative signatures can be identified.