Enhanced methods of ribonucleic acid hybridization

Abstract

This disclosure relates to a method for increasing the hybridization efficiency of a probe and a target RNA in a sample, for example to identify a particular RNA present in the sample. The method includes heating a lysate sample comprising at least one target RNA, such as a tRNA, mRNA or rRNA, at a temperature of about 95° C. for a time sufficient to interfere with secondary structure of the RNA, wherein the time is short enough, such that the RNA in the cell lysate sample are not significantly degraded, and wherein the lysate comprises a cell lysis buffer comprising a chemical denaturant. To detect a target RNA in the lysate, the lysate is contacted with at least one detectable probe, such as a labeled probe, designed to specifically hybridize to the target RNA in the lysate.

We claim: 
     
         1 . A method of increasing the hybridization efficiency of a probe and a target RNA in a cell lysate, comprising:
 heating a cell lysate sample comprising at least one target RNA at a temperature of about 80° C. and about 95° C. for a time sufficient to interfere with secondary structure of the RNA, wherein the time is short enough, such that the RNA in the cell lysate sample are not significantly degraded, and wherein the cell lysate comprises a cell lysis buffer comprising a chemical denaturant;   contacting the cell lysate sample with at least one detectable probe, wherein the at least one probe specifically hybridizes to the target RNA in the cell lysate sample;   
       and detecting hybridization between the probe and the target RNA, wherein the detected hybridization between the probe and the target RNA is increased relative to the hybridization between the probe and the target RNA in the absence of the heating step. 
     
     
         2 . The method of  claim 1 , wherein the lysis buffer comprises a guanidine salt. 
     
     
         3 . The method of  claim 2 , wherein the guanidine salt comprises guanidine isothiocyanate 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the lysis buffer comprises RNAse inactivator. 
     
     
         5 . The method of  claim 4 , wherein the RNAse inactivator comprises a mercaptan. 
     
     
         6 . The method of  claim 5 , wherein the mercaptan comprises ß-mercaptoethanol. 
     
     
         7 . The method of  claim 4 , wherein the RNAse inactivator comprises a metal chelation agent. 
     
     
         8 . The method of any one of  claims 1 - 7 , further comprising, centrifugation of the sample prior to contacting the cell lysate sample with the probe, to remove denatured protein and other cellular debris. 
     
     
         9 . The method of any one of claims,  1 - 8 , wherein the temperature of the sample is maintained at least 65° C. prior to contact with the probe. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the at least one target RNA comprises target ribosomal RNA (rRNA). 
     
     
         11 . The method of any one of  claims 1 - 9 , wherein the at least one target RNA comprises target messenger RNA (mRNA). 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein hybridization between the probe and the target RNA, detects the presence of the target RNA in the sample. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the detectable probe is labeled. 
     
     
         14 . The method of  claim 13 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         15 . The method of  claim 13 , wherein the label comprises a capture moiety. 
     
     
         16 . The method of  claim 15 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the probe is attached to a solid surface. 
     
     
         18 . The method of any one of  claims 1 - 17 , further comprising contacting the sample with a second set of probes, wherein the second set of probes contains at least one second detectable probe that is specific for the target RNA and wherein the individual probes bind to substantially the same region of the RNA as the first set of probes, but wherein the second set of probes do not overlap in sequence identity. 
     
     
         19 . The method of  claim 18 , wherein the second detectable probe is labeled. 
     
     
         20 . The method of  claim 19 , wherein the second detectable probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         21 . The method of  claim 19  or  20 , wherein the label comprises a capture moiety. 
     
     
         22 . The method of  claim 21 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         23 . The method of any one of  claims 18 - 22 , wherein the second detectable probe is attached to a solid surface. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein the sample is obtained from a subject. 
     
     
         25 . The method of any one of  claims 1 - 23 , wherein the sample is an environmental sample. 
     
     
         26 . The method of any one of  claims 1 - 23 , wherein the sample is obtained from a food product. 
     
     
         27 . The method of any one of  claims 1 - 26 , further comprising distinguishing between two or more species of one or more organisms in a sample, comprising:
 contacting the sample with a set of probes, wherein the set of probes contains at least one detectable probe that is specific for a target RNA sequence of each species to be tested, and wherein the individual probes are specific for each species; and   detecting hybridization between one or more of the probes and the RNA, thereby distinguishing between two or more species in the sample.   
     
     
         28 . The method of  claim 27 , wherein the one or more organisms comprise one or more viral pathogens. 
     
     
         29 . The method of  claim 27 , wherein the one or more organisms comprise one or more microorganism. 
     
     
         30 . The method of  claim 29 , wherein the one or more microorganism is selected from the group consisting of bacteria, yeast, parasites, and fungi or a combination thereof. 
     
     
         31 . The method of any one of  claims 28 - 30 , wherein the one or more organisms is a human pathogen. 
     
     
         32 . The method of  claim 31 , wherein the human pathogen comprises bacteria. 
     
     
         33 . The method of any one of  claims 28 - 32 , wherein the detecting hybridization between the probe indicates the presence of the species in the sample. 
     
     
         34 . The method of  claim 33 , wherein the presence of the species in the sample indicates a subject from which the sample was obtained is infected with the organism. 
     
     
         35 . The method of  claim 33 , wherein the presence of the species in the sample indicates that the environmental sample from which the sample was obtained is contaminated with the organism. 
     
     
         36 . The method of  claim 33 , wherein the presence of the species in the sample indicates that the food product from which the sample was obtained is contaminated with the organism.