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

• Current Assignee: Illumina Incorporated
• Original Assignee: Epicentre Technologies Corporation (Illumina Incorporated)
• Inventors: Sooknanan, Roy R.
• Primary Examiner(s): Thomas, David
• Application Number:
  US14/223,788
• Publication Number:
  US 20140235847A1
• Time in Patent Office: 1,254 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

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.

Claims

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 and

d) 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.

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 in said initial sample are from a first eukaryotic organism or species and 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 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).

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 a) both said 28S rRNA molecules and said 18S rRNA molecules, or b) 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 comprises both said 5.8S rRNA and said 5S rRNA molecules, and/or

said at least one rRNA molecules comprises both said 12S and said 16S eukaryotic mitochondrial rRNA molecules, and/or

said at least one rRNA molecule is 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 further comprises RNA molecules derived from at least one prokaryotic species, wherein said prokaryotic RNA molecules comprise prokaryotic rRNA molecules and prokaryotic non-rRNA RNA molecules, and wherein said method further comprises:

providing a composition that comprises affinity-tagged 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 23S, 16S, and 5S prokaryotic rRNA molecules; and

contacting said initial sample with said composition that comprises said prokaryotic affinity-tagged antisense rRNA molecules under conditions such that at least some of said prokaryotic affinity-tagged antisense rRNA molecules and at least some of said prokaryotic rRNA molecules form prokaryotic double-stranded rRNA hybrids; and

contacting said prokaryotic double-stranded rRNA hybrids with said binding matrix in step c) such that at least a portion of said prokaryotic double-stranded rRNA hybrids bind to said binding matrix; and

removing said matrix-bound prokaryotic double-stranded rRNA hybrids, wherein said rRNA-depleted sample further comprises at least a portion of said prokaryotic non-rRNA 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, an environmental sample comprising soil, water, or growth medium, and a biological fluid or specimen.

13. The method of claim 1, wherein said conditions in step b) comprise incubating in the presence of a hybridization buffer at a temperature 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 claim 1, wherein the affinity-tags comprise biotin.

15. The method of claim 1, wherein said affinity-tags are present on said antisense rRNA molecules at a ratio of at least one affinity-tag per every 10 nucleobases of said antisense rRNA molecules.

16. The method of claim 1, 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.

17. The method of claim 10, wherein the composition comprising affinity-tagged antisense rRNA molecules complementary to said at least one eukaryotic rRNA molecule further comprises the composition comprising affinity-tagged antisense rRNA complementary to said at least one prokaryotic rRNA molecule.