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.

For more information and a detailed protocol of L1000 visit lincscloud.org

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.

Phosphoproteomics using the P100 assay  (Jaffe U01)
The P100 assay is a mass spectrometry-based targeted proteomics assay that detects and quantifies a representative set of ~100 phosphopeptide probes that are present in a wide range of cell types and have been demonstrated to be modulated via perturbations. Briefly, phosphopeptides are enriched via an automated protocol from proteins derived from cell pellets that have been perturbed by various reagents. These peptides are mixed with a set of isotopically-labeled internal standards that correspond to the analytes in the P100 assay. This mixture is introduced into a mass spectrometer using ultra-high pressure liquid chromatography (LCMS) and subjected to an analysis method specific for quantification of the P100 analytes. The behavior of more than 1000 unique phosphosites may be imputed from the profiles of the P100 analytes. The resulting profiles are suitable for molecular signature generation, querying of signatures across cell perturbations and types, and modeling of response networks in cellular systems.

For more information and a detailed protocol of P100 and other phosphoproteomic assays being used by LINCS visit lincscloud.org

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.