METHODS, COMPOSITIONS, AND KITS FOR GENERATING rRNA-DEPLETED SAMPLES OR ISOLATING rRNA FROM SAMPLES
First Claim
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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.
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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.
27 Citations
20 Claims
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1. A method of generating a rRNA-depleted sample from an initial sample comprising:
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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; andiii) 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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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.
- View Dependent Claims (16, 17, 18, 19)
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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.
- 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules;
Specification