# US 11,999,992 B2 · Nucleic acid stabilization reagent, kits, and methods of use thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,999,992 B2
- Document ID: US11999992B2
- Status: Granted
- Kind: B2
- Application number: 16/130,634
- Filed: 2018-09-13
- Published: 2019-03-21
- Granted: 2024-06-04
- Earliest claimed: 2016-03-31
- Assignees: Bruker Cellular Analysis, Inc.
- Applicants: Berkeley Lights, Inc.
- Inventors: Jason M. McEwen
- Web page: https://patentgrep.com/patent/US11999992B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- C12N 5/0635: {B lymphocytes}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0645: {Macrophages, e.g. Kuepfer cells in the liver; Monocytes}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Reagents for stabilizing the nucleic acids of a biological cell, compositions, kits and methods of use thereof are described. The stabilization reagents may prepare the nucleic acids within the biological cell for storage and preserve the representative population of the nucleic acids for later isolation and analysis.

## Claims

### Claim 1

A method of stabilizing a population of ribonucleic acids in a biological cell, wherein the biological cell is an animal cell, comprising:
contacting the animal cell with at least one irreversible protein translation inhibitor; at least one ribonucleic acid transcription inhibitor; and at least one electron transport chain agent comprising an electron transport chain inhibitor and/or an electron transport chain decoupling agent, wherein the at least one ribonucleic acid transcription inhibitor is a CDK9 inhibitor, aurethricin, thiolutin, amanitin, and/or triptolide; and/or the electron transport chain agent is rotenone, 2-thenoyltrifluoroacetone, carboxin, cyanide, sodium azide, 2,4 dinitrophenol, dicumarol, and/or carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; and
wherein the contacting is performed for a period of time sufficient to retain the size distribution of the population of ribonucleic acids within the animal cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the animal cell is simultaneously contacted with each of the at least one irreversible protein translation inhibitor, at least one ribonucleic acid transcription inhibitor, and at least one electron transport chain agent.

### Claim 3 (depends on 1)

The method of claim 1, further comprising storing the stabilized animal cell in the presence of each of the at least one irreversible protein translation inhibitor, at least one ribonucleic acid transcription inhibitor, and at least one electron transport chain agent.

### Claim 4 (depends on 3)

The method of claim 3, wherein the step of storing is performed for at least 8 hours and/or at a temperature of 0° C. to 4° C.

### Claim 5 (depends on 1)

The method of claim 1, further comprising: lysing the animal cell by contacting the animal cell with a lysis reagent.

### Claim 6 (depends on 5)

The method of claim 5, further comprising isolating at least a portion of a population of ribonucleic acids released from the lysed animal cell.

### Claim 7 (depends on 6)

The method of claim 6, further comprising analyzing at least one class of ribonucleic acid from the at least a portion of the population of ribonucleic acids released from the lysed animal cell.

### Claim 8 (depends on 7)

The method of claim 7, wherein analyzing includes sequencing the at least one class of ribonucleic acid.

### Claim 9 (depends on 5)

The method of claim 5, wherein the step of lysing further includes washing the animal cell before contacting the biological animal cell with the lysis reagent.

### Claim 10 (depends on 1)

The method of claim 1, wherein the animal cell is a mammalian cell.

### Claim 11 (depends on 1)

The method of claim 1, wherein the animal cell is an immunological cell.

### Claim 12 (depends on 11)

The method of claim 11, wherein the immunological cell is a T cell, a B cell, a NK cell, or a macrophage.

### Claim 13 (depends on 1)

The method of claim 1, wherein more than one of the at least one irreversible protein translation inhibitor; the at least one ribonucleic acid transcription inhibitor;
and the at least one electron transport chain agent are provided in a master mix.

### Claim 14 (depends on 13)

The method of claim 13, wherein all three of the at least one irreversible protein translation inhibitor; the at least one ribonucleic acid transcription inhibitor; and the at least one electron transport chain agent are provided in the master mix.

### Claim 15 (depends on 1)

The method of claim 1, wherein the animal cell is a human cell.

### Claim 16 (depends on 1)

The method of claim 1, wherein the animal cell is a cancer cell.

### Claim 17 (depends on 1)

The method of claim 1, wherein the animal cell is contacted with a second protein translation inhibitor.

### Claim 18 (depends on 1)

The method of claim 1, wherein after the contacting, metabolic gene expression changes are disrupted or blocked in the animal cell.

### Claim 19 (depends on 1)

The method of claim 1, wherein the at least one irreversible protein translation inhibitor is an aminoglycoside antibiotic, D-galactosamine, and/or emetine.

### Claim 20 (depends on 1)

The method of claim 1, wherein the at least one ribonucleic acid transcription inhibitor is a CDK9 inhibitor, aurethricin, thiolutin, amanitin, and/or triptolide; and the electron transport chain agent is rotenone, 2-thenoyltrifluoroacetone, carboxin, cyanide, sodium azide, 2,4 dinitrophenol, dicumarol, and/or carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone.

### Claim 21

A method of stabilizing a population of ribonucleic acids in a biological cell, wherein the biological cell is an animal cell, comprising:
contacting the animal cell with at least one irreversible protein translation inhibitor; at least one ribonucleic acid transcription inhibitor; and at least one electron transport chain agent comprising an electron transport chain inhibitor and/or an electron transport chain decoupling agent, wherein the at least one ribonucleic acid transcription inhibitor is a CDK9 inhibitor, aurethricin, thiolutin, amanitin, and/or triptolide; and/or the electron transport chain agent is rotenone, 2-thenoyltrifluoroacetone, carboxin, cyanide, sodium azide, 2,4 dinitrophenol, dicumarol, and/or carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; and
wherein the contacting is performed for a period of time sufficient to disrupt cellular processes for production and degradation of ribonucleic acids in the population of ribonucleic acids within the animal cell.