Preservation of cell-free nucleic acids

Abstract

A method for preserving and processing cell-free nucleic acids located within a blood sample is disclosed, wherein a blood sample containing cell-free nucleic acids is treated to reduce both blood cell lysis and nuclease activity within the blood sample. The treatment of the sample aids in increasing the amount of cell-free nucleic acids that can be identified and tested while maintaining the structure and integrity of the nucleic acids.

1 . A screening method for the identification of a disease state, comprising the steps of:
 a. contacting a drawn blood sample that includes a plurality of blood cells with a plasma RNA protective agent in an amount and for a time sufficient so that RNA synthesis is inhibited for at least two hours; blood cells of the drawn blood sample are fixed to substantially prevent leaking of cellular RNA into the plasma; any cellular RNA that is within the blood cells at the time of the blood draw is substantially preserved to immobilize the protein expression pattern of the blood cells so that the protein expression pattern of the cells remains substantially the same as at the time of the blood draw; and cell-free RNA that is in the plasma is substantially stabilized against degradation mediated by the combined action of nucleases and proteases;   b. isolating the cell-free RNA from the blood sample; and   c. analyzing (e.g., by quantity, quality, or both) the isolated RNA for the presence, absence, or severity of a disease state.   
     
     
         2 . The method of  claim 1 , wherein the protective agent includes a preservative agent selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1 aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1 aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1 aza-3, 7dioxabicyclo[3.3.0]octane, quaternary adamantine and any combination thereof. 
     
     
         3 . The method of  claim 1 , wherein the protective agent includes diazolidinyl urea. 
     
     
         4 . The method of  claim 1 , wherein the protective agent includes an enzyme inhibitor selected from the group consisting of: diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), glyceraldehydes, sodium fluoride, ethylenediamine tetraacetic acid (EDTA), formamide, vanadyl-ribonucleoside complexes, macaloid, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris (2-carboxyethyl) phosphene hydrochloride, a divalent cation such as Mg +2 , Mn +2 , Zn +2 , Fe +2 , Ca +2 , Cu +2  and any combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the protective agent includes aurintricarboxylic acid and ethylenediamine tetraacetic acid. 
     
     
         6 . The method of  claim 2 , wherein the concentration of the preservative agent prior to the contacting step is about 100 g/l to about 400 g/l. 
     
     
         7 . The method of  claim 2 , wherein the concentration of the preservative agent prior to the contacting step is a concentration at which cross-linking of nucleic acids and proteins is observed, as indicated by agarose gel electrophoresis. 
     
     
         8 . The method of  claim 2 , wherein the amount of the preservative agent is less than about 10 g/l of the blood sample. 
     
     
         9 . The method of  claim 1 , wherein (i) either or both of the isolating or analyzing steps occurs at least 7 days after the blood sample is drawn, (ii) either or both of the isolating or analyzing steps occurs without freezing the blood sample (e.g. to a temperature colder than about −30° C. (more preferably colder than about −70° C.)); or both (i) and (ii). 
     
     
         10 . The method of  claim 1 , wherein the cell-free RNA is mRNA. 
     
     
         11 . The method of  claim 1 , wherein the analyzing step, the isolating step or both includes a step of contacting the nucleic acid with an enzyme, an amplifier or both. 
     
     
         12 . The method of  claim 1 , wherein the contacting step is sufficient so that after a period of at least 7 days from the time the blood sample is drawn, the amount of cell-free RNA present in the blood sample is at least about 90% of the amount of cell-free RNA present in the blood sample at the time the blood sample is drawn. 
     
     
         13 . The method of  claim 1 , wherein the protective agent includes a metabolic inhibitor selected from the group consisting of: glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodium fluoride, K 2 C 2 0 4  and any combination thereof. 
     
     
         14 . The method of  claim 1 , wherein the contacting step is sufficient so that after a period of at least about 7 days from the time the blood sample is drawn, the concentration of cell-free RNA relative to the total nucleic acid in the blood sample that is present is at least about 20 to 50 times the amount of cell-free RNA that would be present in the absence of the contacting step. 
     
     
         15 . The method of  claim 3 , wherein the protective agent includes glyceraldehyde. 
     
     
         16 . The method of  claim 3 , wherein the protective agent includes sodium fluoride. 
     
     
         17 . A composition for the stabilization of RNA within a plasma sample comprising:
 a. from about 0.5% to about 20% by weight one or more nuclease inhibitors;   b. from about 20% to about 30% by weight of one or more preservative agents; and   c. from about 0.05% to about 10% by weight of one or more metabolic inhibitors.   
     
     
         18 . The composition of  claim 17 , wherein the one or more preservative agents includes diazolidinyl urea. 
     
     
         19 . The composition of  claim 17 , wherein the one or more nuclease inhibitors includes aurintricarboxylic acid. 
     
     
         20 . A composition for the stabilization of RNA within a plasma sample comprising:
 a. from about 0.5% to about 2.5% by weight of aurintricarboxylic acid;   b. from about 20% to about 30% by weight diazolidinyl urea;   c. from about 0.05% to about 1.5% by weight sodium fluoride;   d. from about 1% to about 8% by weight glyceraldehyde; and   e. from about 5% to about 15% by weight EDTA.