# US 2019/0169598 A1 · Methods and compositions for isolating small rna

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- Number: US 2019/0169598 A1
- Document ID: US20190169598A1
- Status: Published application
- Kind: A1
- Application number: 16/196,301
- Filed: 2018-11-20
- Published: 2019-06-06
- Earliest claimed: 2003-07-25
- Assignees: Applied Biosystems, LLC
- Applicants: Applied Biosystems, LLC
- Inventors: Richard CONRAD
- Web page: https://patentgrep.com/patent/US20190169598A1

## CPC

- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C07H 1/08: from natural products
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

The present invention concerns the use of methods and compositions for the isolation of small RNA molecules (100 nucleotides or fewer), such as microRNA and siRNA molecules. Such molecules are routinely lost in commonly used isolation procedures and therefore the present invention allows for a much higher level of enrichment or isolation of these small RNA molecules.

## Claims

### Claim at position 1

-53 (canceled)

### Claim 54

A method for isolating small RNA molecules having at most 100 nucleotides or fewer from a sample comprising:
a) lysing cells in a lysing solution to produce a lysate; wherein the lysing solution comprises a chaotropic agent;
b) adding to the lysate an ethanol solution to form a lysate/ethanol mixture;
c) applying the lysate/ethanol mixture to a first solid support and collecting flow-through lysate/ethanol mixture;
d) adding to the flow-through lysate/ethanol mixture of step c) an ethanol solution;
e) applying the lysate/ethanol mixture of step d) to a second solid support; and
f) eluting small RNA molecules from the solid support, wherein the lysate/ethanol mixture applied to the first solid support is between about 20% to about 35% ethanol and wherein the lysate/ethanol mixture applied to the second solid support is between about 35% to about 70% ethanol.

### Claim 55 (depends on 54)

The method of claim 54, wherein the lysing solution comprises a detergent.

### Claim 56 (depends on 55)

The method of claim 55, wherein the concentration of the detergent in the lysing solution is about 0.1% to about 2%.

### Claim 57 (depends on 54)

The method of claim 54 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.

### Claim 58 (depends on 55)

The method of claim 55 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.

### Claim 59 (depends on 56)

The method of claim 56 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.

### Claim 60 (depends on 54)

The method of claim 54, wherein the lysate is extracted with phenol and/or chloroform.

### Claim 61 (depends on 55)

The method of claim 55, wherein the lysate is extracted with phenol and/or chloroform.

### Claim 62 (depends on 57)

The method of claim 57, wherein the lysate is extracted with phenol and/or chloroform.

### Claim 63 (depends on 54)

The method of claim 54, wherein the lysing solution comprises a buffer, preferably in a concentration of about 10 mM to about 300 mM.

### Claim 64 (depends on 60)

The method of claim 60, wherein the lysing solution comprises a buffer, preferably in a concentration of about 10 mM to about 300 mM.

### Claim 65 (depends on 54)

The method of claim 54, further comprising washing the solid support with a first wash solution after applying the lysate to the solid support and optionally washing the solid support with a second wash solution after washing with the first wash solution.

### Claim 66 (depends on 65)

The method of claim 65, wherein the first wash solution comprises a chaotropic agent, preferably wherein the chaotropic agent is a guanidinium salt and the first wash solution further comprises alcohol.

### Claim 67 (depends on 66)

The method of claim 66, wherein the second wash solution comprises alcohol.

### Claim 68 (depends on 54)

The method of claim 54, wherein the small RNA molecules are eluted from the solid support at a temperature of about 60° C. to about 100° C.

### Claim 69 (depends on 57)

The method of claim 57, wherein the small RNA molecules are eluted from the solid support with a low-ionic-strength solution.

### Claim 70 (depends on 54)

The method of claim 54, wherein the solid support comprises silica, in particular glass fiber.

### Claim 71 (depends on 54)

The method of claim 54, wherein the first and the second solid support are made of the same material.