Stabilisation and isolation of extracellular nucleic acids

Abstract

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using an apoptosis inhibitor, preferably a caspase inhibitor, a hypertonic agent and/or a compound according to formula (1) as defined in the claims.

1 . A method for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample by contacting a sample with a caspase inhibitor. 
     
     
         2 . The method according to  claim 1 , comprising contacting the sample with
 a) at least one caspase inhibitor;   b) optionally at least one hypertonic agent which stabilizes the cells comprised in the sample; and   c) optionally at least one compound according to formula 1,   
       
         
           
           
               
               
           
         
         
           wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, more preferred a methyl residue, R2 and R3 are identical or different hydrocarbon residues 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. 
         
       
     
     
         3 . The method according to  claim 1  or  2 , wherein the sample is additionally contacted with an anticoagulant. 
     
     
         4 . The method according to one or more of  claims 1  to  3 , wherein the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample is reduced and/or the degradation of nucleic acids present in the sample is reduced due to the stabilization. 
     
     
         5 . The method according to one or more of  claims 1  to  4 , wherein
 a) the caspase inhibitor has one or more of the following characteristics:
 i) it is a pancaspase inhibitor; and/or 
 ii) it is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK 
 
 and/or 
 b) the hypertonic agent has one or more, preferably two or more of the following characteristics:
 i) it is uncharged; 
 ii) it stabilizes the cells comprised in the sample by inducing cell shrinking; 
 iii) it is cell impermeable; 
 iv) it is water-soluble; 
 v) it is a hydroxylated organic compound; 
 vi) it is a polyol; 
 vii) it is a hydroxy-carbonyl compound; 
 viii) it is a carbohydrate or a sugar alcohol; and/or 
 ix) it is dihydroxyacetone 
 
 and/or 
 c) wherein the compound according to formula 1 has one or more of the following characteristics:
 i) R1, R2 and R3 comprise 1 to 5 carbon atoms; 
 ii) R1, R2 and R3 comprise 1 or 2 carbon atoms; 
 iii) R4 is oxygen; 
 iv) it is a N,N-dialkyl-carboxylic acid amide; 
 v) it is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide; and/or 
 vi) it is N,N-dimethylpropanamide. 
 
 
     
     
         6 . The method according to one or more of  claims 1  to  5 , wherein the sample is additionally contacted with at least one anticoagulant, preferably a chelating agent such as EDTA. 
     
     
         7 . The method according to one or more of  claims 1  to  6 , wherein after the sample has been contacted with the caspase inhibitor, the hypertonic agent, the compound according to formula 1 and/or the anticoagulant, the resulting mixture has one or more of the following characteristics:
 a) it comprises the caspase inhibitor in a concentration selected from at least 0.01 μM, at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at least 3.5 μM; 
 b) it comprises the caspase inhibitor in a concentration range selected from 0.01 μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM or 2.5 μM to 25 μM; 
 c) it comprises the hypertonic agent in a concentration of at least 0.05M, at least 0.1 M, preferably at least 0.25M, more preferably at least 0.5M; 
 d) it comprises the hypertonic agent in a concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or 0.1M to 0.6M; 
 e) it comprises the compound according to formula 1 in a concentration of at least 0.1%, at least 0.5%, at least 1%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%; 
 f) it comprises the compound according to formula 1 in a concentration range selected from 0.1% to 50%, 0.5% to 25%, 0.75% to 20%, 1% to 15% or 1% to 10%; and/or 
 g) it comprises the anticoagulant, preferably a chelating agent, in a concentration range selected from 0.05 mM to 100 mM, 0.05 mM to 50 mM, 0.1 mM to 30 mM, 1 mM to 20 mM or 2 mM to 15 mM. 
 
     
     
         8 . The method according to one or more of  claims 1  to  7 , wherein the sample is for stabilization contacted with:
 a) at least one pancaspase inhibitor as caspase inhibitor, 
 b) at least one hypertonic agent, preferably a hydroxylated organic compound and 
 c) optionally at least one compound according to formula 1, preferably an N,N-dialkyl-carboxylic acid amide, and 
 d) optionally an anticoagulant, preferably a chelating agent, more preferably EDTA, 
 wherein the compounds according to a) to d) are comprised in a stabilising composition. 
 
     
     
         9 . The method according to one or more of  claims 1  to  8 , wherein the sample has one or more of the following characteristics:
 a) it comprises extracellular nucleic acids; 
 b) it is selected from the group consisting of whole blood, samples derived from blood, plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid, ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, semen/seminal fluid, swabs/smears, body fluids, body secretions, nasal secretions, vaginal secretions, wound secretions and excretions and cell culture supernatants; 
 c) it is a cell-depleted or cell-containing body fluid; 
 d) it is selected from whole blood, plasma and/or serum; and/or 
 e) it is whole blood. 
 
     
     
         10 . The method according to one or more of  claims 1  to  9 , wherein stabilization of the extracellular nucleic acid population is achievable without refrigeration, preferably at room temperature, for a time period selected from
 a) at least two days; 
 b) at least three days; 
 c) at least one day to three days; 
 d) at least one day to six days; and/or 
 e) at least one day to seven days. 
 
     
     
         11 . The method according to one or more of  claims 1  to  10 , wherein
 a) the one or more stabilising agents and optionally further additives are comprised in a stabilising composition and wherein the volumetric ratio of the stabilising composition to the specified volume of the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5; 
 b) the stabilized sample is subjected to a nucleic acid analysis and/or detection method; 
 c) extracellular nucleic acids are isolated from the stabilized sample; 
 d) extracellular nucleic acids are isolated from the stabilized sample and the isolated nucleic acids are analysed and/or detected; 
 e) cells comprised in the stabilized sample are removed; 
 f) cells comprised in the stabilized sample are removed prior to performing an isolation, analysis and/or detection step; 
 g) a nucleic acid isolation step is performed after a stabilization period as defined in  claim 10 ; 
 h) (i) the stabilized sample, (ii) the stabilized sample from which cells have been removed and/or (iii) cells removed from the sample are stored; 
 i) cells that were removed from the stabilized sample are discarded; and/or 
 j) nucleic acids are isolated from cells that were removed from the stabilized sample. 
 
     
     
         12 . The method according to one or more of  claims 1  to  11 , for stabilizing an extracellular nucleic acid population comprised in a blood sample, comprising contacting the blood sample with a caspase inhibitor and an anticoagulant, wherein the release of genomic DNA from cells contained in the blood sample into the cell-free portion of the blood sample is reduced and the degradation of nucleic acids present in the sample is reduced due to the stabilization. 
     
     
         13 . A method for isolating extracellular nucleic acids from a biological sample, preferably a blood sample, comprising the steps of:
 a) stabilizing the extracellular nucleic acid population in the sample according to the method defined in one or more of  claims 1  to  12 ; and   b) isolating extracellular nucleic acids.   
     
     
         14 . The method according to  claim 13 , comprising one or more of the following steps:
 i) optionally removing cells from the cell-containing sample between step a) and step b);   ii) performing one or more of the steps b) to j) as defined in  claim 11 ; and/or   iii) isolating extracellular nucleic acids from the sample in step b) of  claim 13  using an isolation method selected from the group comprising extraction, solid-phase extraction, isolation methods using a nucleic acid binding solid phase, isolation methods using a silica material, isolation methods that are based on the use of a solid phase comprising anionic exchange groups; magnetic particle-based purification, phenol-chloroform extraction, alcohol and/or chaotropic agent(s) based nucleic isolation method, chromatography, anion-exchange chromatography, anion exchange particle-based isolation, electrophoresis, filtration, precipitation, target nucleic acid specific isolation methods and combinations thereof.   
     
     
         15 . The method according to  claim 13  or  14 , wherein the isolated nucleic acids are in a further step c) processed and/or analysed and preferably are:
 i) modified; 
 ii) contacted with at least one enzyme; 
 iii) amplified; 
 iv) reverse transcribed; 
 v) cloned; 
 vi) sequenced; 
 vii) contacted with a probe; 
 viii) detected; 
 ix) quantified; and/or 
 ix) identified. 
 
     
     
         16 . The method according to one or more of  claims 13  to  15 , wherein
 a) the extracellular nucleic acid population that is isolated from the cell-free portion of the sample and/or that is obtained after isolation in step b) of  claim 13 , has one or more of the following characteristics:
 i) it is comprised as a portion in the total nucleic acid that is isolated; 
 ii) it predominantly comprises DNA; 
 iii) it predominantly comprises RNA; 
 iv) it comprises circulating extracellular nucleic acids; 
 v) it comprises disease related nucleic acids; 
 vi) it comprises tumor-associated or tumor-derived nucleic acids; 
 vii) it comprises inflammation related nucleic acids: 
 viii) it comprises fetal nucleic acids; 
 ix) it comprises viral nucleic acids; 
 x) it comprises pathogen nucleic acids; 
 xi) it comprises mammalian extracellular nucleic acids; and/or 
 xii) it is a mixture of DNA and RNA; 
 
 and/or 
 b) the extracellular nucleic acid that is analysed and/or further processed, preferably detected, in step c), has one or more of the following characteristics:
 i) it is DNA; 
 ii) it is RNA; 
 iii) it is a circulating extracellular nucleic acid; 
 iv) it comprises disease related nucleic acids; 
 v) it comprises tumor-associated or tumor-derived nucleic acids; 
 vi) it comprises inflammation related nucleic acids: 
 vii) it is a fetal nucleic acid; 
 viii) it is a viral nucleic acid; 
 ix) it is a pathogen nucleic acid; 
 x) it is a mammalian extracellular nucleic acid; and/or 
 xi) it is a mixture of DNA and RNA; 
 
 
     
     
         17 . A composition suitable for stabilizing the extracellular nucleic acid population in a biological sample, preferably a blood sample, comprising
 a) at least one caspase inhibitor,   and comprising at least one further compound selected from b), c) and d), wherein   b) is at least one hypertonic agent suitable for stabilizing cells comprised in the sample, preferably is at least one hydroxylated organic compound;   c) is at least one compound according to formula 1,   
       
         
           
           
               
               
           
         
         
           wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, more preferred a methyl residue, R2 and R3 are identical or different hydrocarbon residues 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; 
         
         d) is at least one anticoagulant, preferably a chelating agent. 
       
     
     
         18 . The composition according to  claim 17 , comprising at least one caspase inhibitor and at least one anticoagulant. 
     
     
         19 . The composition according to  claim 17  or  18 , having one or more of the following characteristics:
 a) it is capable of reducing the release of genomic DNA from cells contained in the 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 sample; 
 c) the caspase inhibitor has one or more of the characteristics as defined in  claim 5 ; 
 d) it comprises at least one hypertonic agent as defined in  claim 5 ; 
 e) it comprises at least one compound according to formula 1 as defined in  claim 5 ; 
 f) when mixed with a biological sample, preferably blood, plasma or serum, the resulting mixture comprises the caspase inhibitor, the hypertonic agent, the compound according to formula 1 and/or the chelating agent in a concentration as defined in  claim 7 ; 
 g) it is provided in a solid form; 
 h) it is provided in a liquid form; and/or 
 i) it is capable of stabilizing the extracellular nucleic acid population contained in said sample at room temperature for at least 3 days, preferably at least 6 days. 
 
     
     
         20 . The composition according to one or more of  claims 17  to  19 , wherein the stabilizing composition is provided as mixture with a biological sample and wherein said sample has one or more of the following characteristics:
 a) it comprises extracellular nucleic acids; 
 b) it is selected from the group consisting of whole blood, plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid, ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, semen/seminal fluid, swabs/smears, body fluids, body secretions, nasal secretions, vaginal secretions, wound secretions and excretions and cell culture supernatants; 
 c) it is a cell-depleted or cell containing body fluid; 
 d) it is selected from whole blood, plasma and/or serum; and/or 
 e) it is whole blood. 
 
     
     
         21 . The composition according to  claim 20 , wherein volumetric ratio of the stabilising composition to the specified volume of the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5.