METHODS, COMPOSITIONS, AND KITS FOR GENERATING rRNA-DEPLETED SAMPLES OR ISOLATING rRNA FROM SAMPLES

Abstract

The present invention provides methods, compositions, and kits for generating rRNA-depleted samples and for isolating rRNA from samples. In particular, the present invention provides compositions comprising affinity-tagged antisense rRNA molecules corresponding to substantially all of at least one rRNA molecule (e.g., 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S prokaryotic rRNA molecules) and methods for using such compositions to generate rRNA-depleted samples or to isolate rRNA molecules from samples.

1 . A method of generating a rRNA-depleted sample from an initial sample comprising:
 a) providing;
 i) an initial sample comprising RNA molecules derived from at least one eukaryotic organism or species, wherein said RNA molecules comprise rRNA molecules and non-rRNA RNA molecules; 
 ii) a composition comprising antisense rRNA molecules complementary to substantially all of the sequence of at least one rRNA molecule selected from the group consisting of: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules and 12S and 16S eukaryotic mitochondrial rRNA molecules, wherein said antisense rRNA molecules comprise affinity-tags at a ratio of at least one affinity-tag per every 10 nucleobases of said antisense rRNA molecules; and 
 iii) a binding matrix comprising affinity-tag-binding molecules; 
   b) contacting said initial sample with said composition comprising antisense rRNA molecules under conditions such that at least some of said affinity-tagged antisense rRNA molecules and at least some of said rRNA molecules form double-stranded rRNA hybrids, thereby generating a treated sample;   c) contacting said treated sample with said binding matrix under conditions wherein the ratio of said affinity-tag-binding molecules to said affinity tags present in the antisense rRNA molecules used in step b) is at least 8 to 1, such that at least a portion of said double-stranded rRNA hybrids bind to said binding matrix and are removed from said treated sample, thereby generating an rRNA-depleted sample, wherein said rRNA-depleted sample comprises at least a portion of said non-rRNA RNA molecules present in said initial sample and is substantially depleted or free of rRNA molecules that exhibit sequences exhibited by said at least one rRNA molecule.   
     
     
         2 . The method of  claim 1 , wherein said at least one eukaryotic organism or species is selected from the group consisting of a human, an animal, a plant, and a fungal organism or species. 
     
     
         3 . The method of  claim 2 , wherein said RNA molecules in said initial sample and said at least one rRNA molecule are derived from the same eukaryotic organism or species. 
     
     
         4 . The method of  claim 1 , wherein said RNA molecules are from a first eukaryotic organism or species, and wherein said at least one rRNA molecule is from a second eukaryotic organism or species. 
     
     
         5 . The method of  claim 4 , wherein one of said first or second organisms or species is  Homo sapiens , and wherein the other of said first or second organisms or species is a non-human mammal. 
     
     
         6 . The method of  claim 1 , wherein said affinity-tags are associated with at least one nucleobase selected from the group consisting of: adenine (A), cytosine (C), guanine (G) and uracil (U); and
 wherein said affinity-tags are present on said antisense rRNA molecules at a ratio of at least one affinity-tag per every 8 nucleobases of said antisense rRNA molecules; and/or   wherein the ratio of said affinity-tag-binding molecules to said affinity tags during said contacting in step b) is at least 10 to 1.   
     
     
         7 . The method of  claim 1 , wherein the number of molecules that exhibit the sequence of said at least one rRNA molecule in said rRNA-depleted sample is depleted by 99.0% relative to the number of molecules that exhibit the sequence of said at least one rRNA molecule in said initial sample. 
     
     
         8 . The method of  claim 1 , wherein said at least one rRNA molecule includes either both said 28S rRNA molecules and said 18S rRNA molecules, or both said 25S or 26S rRNA molecules and said 16S rRNA molecules. 
     
     
         9 . The method of  claim 1 , wherein said at least one rRNA molecule additionally includes both said 5.8 S rRNA and said 5S rRNA molecules, and/or
 wherein said at least one rRNA molecule additionally includes both said 12S and said 16S eukaryotic mitochondrial rRNA molecules, and/or   wherein said at least one rRNA molecule is additionally selected from the group consisting of 16S, 23S, 4.5S and 5S eukaryotic chloroplast rRNA molecules.   
     
     
         10 . The method of  claim 1 , wherein said initial sample comprising RNA molecules derived from a eukaryotic organism or species additionally comprises RNA molecules derived from at least one prokaryotic species, wherein said prokaryotic RNA molecules comprise rRNA molecules and non-rRNA RNA molecules, and wherein said method further comprises:
 providing a composition that additionally comprises affinity-tagged antisense rRNA molecules complementary to substantially all of the sequence of at least one rRNA molecule selected from the group consisting of 23S, 16S, and 5S prokaryotic rRNA molecules; and   contacting said initial sample with said composition that additionally comprises prokaryotic affinity-tagged antisense rRNA molecules under conditions such that at least some of said affinity-tagged antisense rRNA molecules and at least some of said prokaryotic rRNA molecules form double-stranded rRNA hybrids thereby generating a treated sample; and   contacting said treated sample with said binding matrix in step c) under conditions wherein said rRNA-depleted sample also comprises at least a portion of said non-rRNA prokaryotic RNA molecules present in said initial sample and is substantially depleted or free of prokaryotic rRNA molecules that exhibit sequences exhibited by said at least one prokaryotic rRNA molecule.   
     
     
         11 . The method of  claim 1 , wherein at least a portion of said RNA molecules in said initial sample are fragmented. 
     
     
         12 . The method of  claim 1 , wherein said RNA molecules in said initial sample comprise RNA extracted, isolated, or purified from a source selected from the group consisting of: a tissue sample, a cell sample, a paraffin-embedded sample, a paraffin-embedded formalin-fixed (FFPE) sample, and an environmental sample consisting of soil, water, growth medium, or a biological fluid or specimen. 
     
     
         13 . The method of  claims 1 , wherein said conditions in step b) comprise incubating in the presence of a hybridization buffer at a temperate of about 60-75° C. for a first time period and incubating at about room temperature for a second time period, and wherein said binding matrix in step c) comprises a plurality of individual particles or a binding column, and wherein said conditions in step c) include at least occasional mixing at room temperature and/or at 35-60° C. 
     
     
         14 . The method of  claims 1 , wherein the affinity-tag comprises biotin. 
     
     
         15 . A composition comprising antisense rRNA molecules complementary to substantially all of the sequence exhibited by at least one rRNA molecule selected from the group consisting of: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, 16S, 23S, 4.5S and 5S eukaryotic chloroplast rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules, wherein said antisense rRNA molecules comprise affinity-tags at a ratio of at least one affinity-tag per every 10 nucleobases of said antisense rRNA molecules, or wherein said antisense rRNA molecules comprise affinity-tags at a ratio of at least one affinity-tag per every 8 nucleobases of said antisense rRNA molecules. 
     
     
         16 . The composition of  claim 15 , wherein said antisense rRNA molecules comprise multiple different anti-sense rRNA molecules complementary to rRNA molecules selected from the groups consisting of: at least four rRNA molecules selected from 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules; two rRNA molecules consisting of 12S and 16S eukaryotic mitochondrial rRNA molecules; four rRNA molecules consisting of 16S, 23S, 4.5S and 5S eukaryotic chloroplast rRNA molecules; and three rRNA molecules consisting of 23S, 16S, and 5S prokaryotic rRNA molecules. 
     
     
         17 . The composition of  claim 15 , additionally comprising RNA molecules derived from at least one organism or species, wherein said RNA molecules comprise rRNA molecules and non-rRNA RNA molecules, wherein at least some of said affinity-tagged antisense rRNA molecules in said composition are present as double-stranded hybrids with at least some of said rRNA molecules. 
     
     
         18 . The composition of  claim 17 , wherein the said RNA molecules comprise RNA molecules derived from multiple organisms or species. 
     
     
         19 . The composition of  claim 17 , further comprising a binding matrix comprising affinity-tag-binding molecules, wherein the ratio of said affinity-tag-binding molecules to said affinity tags present in the antisense rRNA molecules used in step b) is at least 8 to 1, or wherein the ratio of said affinity-tag-binding molecules to said affinity tags present in the antisense rRNA molecules used in step b) is at least 10 to 1. 
     
     
         20 . A composition comprising an rRNA-depleted sample comprising non-rRNA RNA molecules, wherein, compared to a sample that is not rRNA-depleted, said composition is substantially free of rRNA molecules that exhibit the sequence of at least one rRNA molecule selected from the group consisting of: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules; 12S and 16S eukaryotic mitochondrial rRNA molecules; 16S, 23S, 4.5S and 5S eukaryotic chloroplast rRNA molecules; and 23S, 16S, and 5S prokaryotic rRNA molecules.