Detection of Target Nucleic Acids in a Cellular Sample

Abstract

Methods of assaying cells of a cellular sample for the presence of a target nucleic acid are provided. Aspects of the methods include evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid. Also provided are devices and kits that find use in practicing the methods described herein.

1 . A method of assaying a cellular sample for the presence of a target nucleic acid, the method comprising:
 (i) contacting the cellular sample with a fixation reagent and a permeabilization reagent to produce a fixed/permeabilized cellular sample;   (ii) contacting the fixed/permeabilized cellular sample with an aqueous post-fixation reagent to produce a rehydrated/fixed cellular sample;   (iii) contacting the rehydrated/fixed cellular sample with a nuclease inhibitor; and   (iv) evaluating the nuclease inhibitor-contacted rehydrated/fixed cellular sample for the presence of the target nucleic acid.   
     
     
         2 . The method according to  claim 1 , wherein the target nucleic acid is a ribonucleic acid. 
     
     
         3 . The method according to  claim 2 , wherein the ribonucleic acid is an mRNA, a microRNA, a fusion gene transcript, or a splice variant. 
     
     
         4 . The method according to  claim 2 , wherein the nuclease inhibitor is an RNase inhibitor. 
     
     
         5 . The method according to  claim 1 , wherein the method further comprises contacting the cellular sample with a protein detection reagent prior to step (i) and/or prior to step (iv). 
     
     
         6 . The method according to  claim 5 , wherein the protein detection reagent comprises a labeled binding member that specifically binds to a target protein. 
     
     
         7 . The method according to  claim 6 , wherein the labeled binding member comprises an antibody or binding fragment thereof. 
     
     
         8 . The method according to  claim 6 , wherein the label is a metal. 
     
     
         9 . The method according to  claim 6 , wherein the evaluating comprises measuring the amount of label using mass cytometry. 
     
     
         10 . The method according to  claim 1 , wherein the evaluating comprises contacting the nuclease inhibitor-contacted rehydrated/fixed cellular sample with a nucleic acid detection agent. 
     
     
         11 . The method according to  claim 10 , wherein the nucleic acid detection agent comprises a signal producing system comprising a labeled nucleic acid probe. 
     
     
         12 . The method according to  claim 11 , wherein the label is a metal. 
     
     
         13 . The method according to  claim 12 , wherein the evaluating comprises measuring the amount of label using mass cytometry. 
     
     
         14 . The method according to  claim 11 , wherein the signal producing system comprises a signal amplification component. 
     
     
         15 . The method according to  claim 14 , wherein the signal amplification component is a branched nucleic acid. 
     
     
         16 . The method according to  claim 1 , wherein the evaluating comprises flow cytometrically analyzing the nuclease inhibitor-contacted rehydrated/fixed cellular sample. 
     
     
         17 . The method according to  claim 1 , wherein the method comprises storing the nuclease inhibitor-contacted rehydrated/fixed cellular sample for a period of time prior to the evaluating step. 
     
     
         18 . The method according to  claim 17 , wherein the nuclease inhibitor-contacted rehydrated/fixed cellular sample is stored in an aqueous buffer comprising a nuclease inhibitor. 
     
     
         19 . The method according to  claim 18 , wherein the nuclease inhibitor-contacted rehydrated/fixed cellular sample is stored at a temperature below 0° C. 
     
     
         20 . The method according to  claim 1 , wherein the cellular sample is contacted with a stimulating agent prior to contact with the fixation reagent. 
     
     
         21 .- 79 . (canceled)