Method of screening a plurality of single secreting cells for functional activity

Abstract

This invention generally relates to methods, devices and kits for screening a plurality of single secreting cells for functional activity of the secreted molecules by measuring the amount of reporter gene mRNA produced in one or more reporter cells in response to the secreted molecules.

1 . A method of measuring a response of a reporter cell to a binding of a ligand to a receptor comprising the steps of:
 placing a plurality of ligands into microwells;   placing one or more reporter cells of one or more types into the microwells occupied by the ligands;   allowing the ligands to interact with a receptor of the reporter cells;   lysing the reporter cells in the microwells;   capturing mRNA from one or more of reporter genes of the reporter cells and mRNA from one or more housekeeping genes of the reporter cells from the microwells with an oligonucleotide capture support containing mRNA capture oligos where the mRNA capture oligos comprise a DNA tag unique to each microwell and a random code;   converting captured mRNA into cDNA incorporating the DNA tag and random code;   sequencing the tagged cDNA by next generation sequencing (NGS);   examining the sequenced cDNA from the one or more reporter genes of the reporter cells;   using the DNA tags and random codes to compute an Absolute Copy Number (ACN) of the reporter genes and an ACN of housekeeping genes for the reporter cells in each well;   using a ratio of the ACN of the reporter genes to the ACN of the housekeeping genes to compute a Normalized Copy Number (NCN) of the reporter genes for the reporter cells in each well;   examining the mRNA sequences encoding the reporter genes from the reporting cells in each well; and   for each well, associating the mRNA sequences encoding the reporter genes from the reporter cells with the NCN of the mRNA from the reporter genes.   
     
     
         2 . The method of  claim 1  wherein the number of nucleotides in the random code is more than 4 nucleotides. 
     
     
         3 . The method of  claim 1  wherein the receptor is a membrane bound protein permanently bound to a lipid bilayer of the reporter cell, a peripheral membrane protein temporarily associated with the lipid bilayer or integral membrane protein, or a lipid-anchored protein bound to the lipid bilayer through lipidated amino acid residues. 
     
     
         4 . The method of  claim 1 , wherein the volume of the microwells is between 10 and 1000 picoliters. 
     
     
         5 . The method of  claim 1 , wherein the number of nucleotides in the DNA tag is more than 6 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein the number of reporter cells deposited into said microwells is between 1 and 500 cells per microwell.