Stabilization and isolation of extracellular nucleic acids

Patent metadata

Field	Value
Publication number	US-2016281133-A1
Application number	US-201414777882-A
Country	US
Kind code	A1
Filing date	Mar 18, 2014
Priority date	Mar 18, 2013
Publication date	Sep 29, 2016
Grant date	—

Abstract

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using butanamide.

First claim

1 . A container for collecting a cell-containing biological sample, wherein the container comprises butanamide and at least one further additive selected from the group consisting of
an apoptosis inhibitor, an anticoagulant and a compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, preferably a C1-C2 alkyl residue, R2 and R3 are identical or different and are selected from a hydrogen residue and a hydrocarbon residue with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen, sulphur or selenium residue, preferably R4 is oxygen.

2 . The container according to claim 1 , wherein the container comprises a composition suitable for stabilizing a cell-containing biological sample wherein the composition comprises butanamide and at least one further additive selected from the group consisting of
an apoptosis inhibitor, an anticoagulant and a compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, preferably a C1-C2 alkyl residue, R2 and R3 are identical or different and are selected from a hydrogen residue and a hydrocarbon residue with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen, sulphur or selenium residue, preferably R4 is oxygen.

3 . The collection container of claim 2 , wherein the stabilizing composition comprises an apoptosis inhibitor which is a caspase inhibitor.

4 . The collection container according to one or more of claims 1 to 3 , wherein the container comprises:
a) butanamide in concentration so that when the cell-containing biological sample is collected into the container, the concentration of butanamide in the resulting mixture is at least 0.25% (w/v), at least 0.3% (w/v), at least 0.4% (w/v), at least 0.5% (w/v), at least 0.75% (w/v) or at least 1% (w/v), at least 1.5% (w/v), at least 1.75% (w/v), at least 2% (w/v) or at least 2.5% (w/v) and wherein preferably, the resulting mixture comprises butanamide in a concentration that lies in the range of 0.25% (w/v) to 15% (w/v), 0.5% (w/v) to 12.5% (w/v), 0.75% (w/v) to 10% (w/v), 0.8% (w/v) to 9% (w/v), 0.9% (w/v) to 8% (w/v), 1% (w/v) to 7% (w/v), 1.1% (w/v) to 6% (w/v), 1.2% (w/v) to 6% (w/v), 1.3% (w/v) to 5.5% (w/v), 1.4% (w/v) to 5.25% (w/v), 1.5% (w/v) to 5% (w/v), 1.75% (w/v) to 4.75% (w/v), 2.0% (w/v) to 4.5% (w/v), 2.2% (w/v) to 4.25% (w/v), 2.3% (w/v) to 4% (w/v), 2.4% (w/v) to 3.75% (w/v) or 2.5% (w/v) to 3.5% (w/v); and
b) at least one caspase inhibitor in a concentration so that when the cell-containing biological sample is collected into the container, the concentration of the caspase inhibitor in the resulting mixture is at least 0.01 μM, at least 0.1 μM, at least 0.25 μM, at least 0.5 μM, at least 0.6 μM, at least 0.7 μM, at least 0.8 μM, at least 0.9 μM or at least 1 μM and wherein preferably, the resulting mixture comprises the caspase inhibitor in a concentration that lies in the range of 0.01 μM to 100 μM, 0.1 μM to 75 μM, 0.25 μM to 50 μM, 0.5 μM to 40 μM, 0.6 μM to 30 μM, 0.7 μM to 35 μM, 0.8 μM to 30 μM, 0.9 μM to 25 μM, 1 μM to 20 μM, 1.1 μM to 17.5 μM, 1.25 μM to 15 μM or 1.5 μM to 12.5 μM.

5 . The collection container according to one or more of claims 1 to 4 , comprising
a) butanamide;
b) at least one pancaspase inhibitor;
c) optionally at least one compound according to formula 1, preferably a N,N-dialkyl-carboxylic acid amide, more preferred a N,N-dialkylpropanamide; and
d) optionally an anticoagulant, preferably a chelating agent, more preferably EDTA.

6 . The collection container according to claim 5 , wherein the pancaspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK, and more preferably is Q-VD-OPh.

7 . The collection container according to one or more of claims 1 to 6 , wherein the container additionally comprises
c) a compound according to formula 1.

8 . The collection container according to claim 7 , wherein the compound according to formula 1 has one or more of the following characteristics:
i) it is a tertiary carboxylic acid amide; ii) it is a N,N-dialkyl-carboxylic acid amide; iii) it is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylpropanamide; iv) it is a N,N-dialkylpropanamide; v) it is N,N-dimethylpropanamide; vi) it is a non-toxic compound; and/or vii) it is comprised in a concentration so that when the cell-containing biological sample is collected into the container, the concentration of the compound according to formula 1 in the resulting mixture is at least 0.1%, at least 0.5%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5% and wherein preferably, the resulting mixture comprises the compound according to formula 1 in a concentration that lies in the range of 0.1% to 30%, 0.25% to 20%, 0.5% to 15%, 0.7% to 10%, 0.8% to 7.5%, 0.9% to 6% or 1% to 5%.

9 . The collection container according to claim 7 or 8 , comprising a caspase inhibitor, butanamide and N,N-dimethylpropanamide.

10 . The collection container according to one or more of claims 1 to 9 , wherein the container additionally comprises an anticoagulant.

11 . The collection container according to claim 10 , wherein the container comprises a chelating agent, preferably EDTA, in a concentration so that when the cell-containing biological sample is collected into the container, the concentration of the chelating agent in the resulting mixture lies in a concentration range selected from 0.05 mM to 100 mM, 0.1 mM to 50 mM, 0.5 mM to 30 mM, 1 mM to 20 mM, 1.5 mM to 15 mM or 2 mM to 15 mM.

12 . The collection container according to one or more of claims 1 to 11 , wherein the container is evacuated.

13 . The collection container according to one or more of claims 2 to 12 , wherein the stabilizing composition comprised in the container has one or more of the following characteristics:
a) it is capable of stabilizing cells and reducing the release of genomic DNA from cells contained in the cell-containing biological sample into the cell-free portion of the sample;
b) it is capable of reducing the degradation of nucleic acids, in particular genomic DNA, present in the stabilized sample;
c) it is capable of reducing or preventing the contamination of the extracellular DNA population comprised in the biological sample with genomic DNA originating from cells contained in the stabilized sample;
d) it is capable of reducing or preventing the contamination of the extracellular nucleic acid population comprised in the biological sample with intracellular nucleic acids originating from cells contained in the stabilized sample;
e) the stabilization composition does not comprise additives in a concentration wherein said additives would induce or promote cell lysis;
f) the stabilization composition does not comprise a cross-linking agent that induces protein-DNA and/or protein-protein crosslinks;
g) the stabilization composition does not comprise formaldehyde, formaline, paraformaldehyde or a formaldehyde releaser;
h) the stabilization composition does not comprise a toxic agent and/or
i) it is capable of stabilizing extracellular nucleic acid population comprised

Assignees

• Qiagen Gmbh

Inventors

• Wyrich Ralf
• Voss Thorsten
• Grölz Daniel

Classifications

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Q1/6806 Primary

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