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Methods, compositions, and kits for generating rRNA-depleted samples or isolating rRNA from samples

  • US 9,745,570 B2
  • Filed: 03/24/2014
  • Issued: 08/29/2017
  • Est. Priority Date: 08/14/2009
  • Status: Active Grant
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 at least 95% of a full-length 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 eight affinity-tags per every 100 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 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 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 andd) removing said matrix-bound double-stranded rRNA hybrids 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 of rRNA molecules that exhibit sequences exhibited by said at least one rRNA molecule.

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