Nucleic acid stabilization reagent, kits, and methods of use thereof
Inventors
Assignees
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Publication Number
US-11999992-B2
Publication Date
2024-06-04
Expiration Date
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.
Core Innovation
The invention describes a method of stabilizing a population of ribonucleic acids in a biological cell, wherein the biological cell is an animal cell. The method comprises 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.
The ribonucleic acid transcription inhibitor is selected from CDK9 inhibitor, aurethricin, thiolutin, amanitin, and/or triptolide. The electron transport chain agent is selected from rotenone, 2-thenoyltrifluoroacetone, carboxin, cyanide, sodium azide, 2,4 dinitrophenol, dicumarol, and/or carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone.
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, or sufficient to disrupt cellular processes for production and degradation of ribonucleic acids in the population of ribonucleic acids within the animal cell.
The document also extends nucleic acid stabilization into microfluidic devices using a sequestration-pen chamber/fluidic-channel enclosure that provides diffusion-only exchange and diffusion-dominant isolation from flowing media, with optional onboard processing including lysis, nucleic acid capture on matrices/beads, and on- or off-chip analysis.
Claims Coverage
The consolidated claims coverage includes two independent claims directed to stabilizing a population of ribonucleic acids in an animal cell. Across the independent claims, the core inventive structure requires three classes of inhibitors/agents and a contacting duration tied to either retention of RNA size distribution or disruption of RNA production and degradation processes. Two functional alternatives and the specified agent selections are recited.
Three-component contacting for stabilizing ribonucleic acids
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.
Specific ribonucleic acid transcription inhibitor selection
The at least one ribonucleic acid transcription inhibitor is a CDK9 inhibitor, aurethricin, thiolutin, amanitin, and/or triptolide.
Specific electron transport chain agent selection
The electron transport chain agent is rotenone, 2-thenoyltrifluoroacetone, carboxin, cyanide, sodium azide, 2,4 dinitrophenol, dicumarol, and/or carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone.
Time sufficient to retain ribonucleic acid size distribution
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.
Time sufficient to disrupt production and degradation processes
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.
Both independent claims cover stabilizing a population of ribonucleic acids in an animal cell by contacting with an irreversible protein translation inhibitor, a specified ribonucleic acid transcription inhibitor, and a specified electron transport chain agent, with the contacting duration defined by retention of RNA size distribution or disruption of cellular production and degradation processes.
Stated Advantages
Retains the size distribution of the population of ribonucleic acids within the animal cell.
Disrupts cellular processes for production and degradation of ribonucleic acids in the population within the animal cell.
Reduced expression variability.
Documented Applications
Stabilized cells are suitable for later isolation and sequencing/hybridization of ribonucleic acids.
Stabilizing a population of ribonucleic acids in an animal cell for preservation of ribonucleic acid size distribution.
Microfluidic devices using a sequestration-pen chamber/fluidic-channel enclosure with diffusion-dominant isolation from flowing media.
Microfluidic/nanofluidic OptoSelect (OEP/NanoPen) device implementations, including priming/perfusion, adding a stabilization reagent into microfluidic chambers, storing at 4°C overnight, and then exporting to lysis and sequencing for reduced expression variability.
Onboard processing in microfluidic devices, including lysis, nucleic acid capture on matrices/beads, and on- or off-chip analysis.
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