Nucleic acid stabilization reagent, kits, and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11999992-B2
Application number	US-201816130634-A
Country	US
Kind code	B2
Filing date	Sep 13, 2018
Priority date	Mar 31, 2016
Publication date	Jun 4, 2024
Grant date	Jun 4, 2024

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.

First claim

What is claimed is:

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.

2. 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.

3. 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.

4. 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.

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

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

7. 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.

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

9. 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.

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

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

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

13. 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.

14. 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.

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

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

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

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

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

20. 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.

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.

Assignees

• Berkeley Lights Inc
• Bruker Cellular Analysis Inc

Inventors

• Mcewen Jason M

Classifications

• C12N5/0646

  Natural killers cells [NK], NKT cells · CPC title

• C12N5/0645

  Macrophages, e.g. Kuepfer cells in the liver; Monocytes · CPC title

• C12N5/0636

  T lymphocytes · CPC title

• C12N5/0635

  B lymphocytes · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title