Method for isolating rna with high yield

Abstract

A phenol-free method for isolating a nucleic acid from a sample is provided, said method comprising the following steps: a) adding a precipitation buffer to a sample to prepare an acidic precipitation mixture wherein said precipitation buffer comprises a metal cation precipitant and a buffering agent, has a pH value of 4.0 or less and does not comprise an organic solvent selected from aprotic polar solvents and protic solvents and wherein the acidic precipitation mixture comprises the metal cation precipitant in a concentration of less than 200 mM and precipitating proteins; b) separating the precipitate from the supernatant, wherein the supernatant comprises small RNA having a length of less than 200 nt and large RNA having a length of at least 1000 nt; and c) isolating a nucleic acid from the supernatant. The present method allows to avoid the use of organic solvents during protein precipitation. Also provided is a precipitation buffer.

1 . A phenol-free method for isolating a nucleic acid from a sample, said method comprising:
 a) combining a precipitation buffer with a sample to prepare an acidic precipitation mixture,
 wherein said precipitation buffer comprises a metal cation precipitant and a buffering agent, has a pH value of 4.0 or less, and does not comprise an organic solvent selected from aprotic polar solvents and protic solvents, wherein the protic solvents are linear or branched C1-C5 alcohols, and wherein the acidic precipitation mixture comprises the metal cation precipitant in a concentration of less than 200 mM and precipitating proteins; 
   b) separating the precipitate from the supernatant, wherein the supernatant comprises small RNA having a length of less than 200 nt and large RNA having a length of at least 1000 nt; and   c) isolating a nucleic acid from the supernatant.   
     
     
         2 . The method according to  claim 1 , wherein the metal cation precipitant is selected from Zn 2+ , Cu 2+  and Al 3+ . 
     
     
         3 . The method according to  claim 1 , having one or more of the following characteristics:
 i) the precipitation mixture provided in step a) comprises the metal cation precipitant in a concentration of 175 mM or less, 170 mM or less, 165 mM or less, 160 mM or less, 155 mM or less, 150 mM or less, 145 mM or less or 140 mM or less;   ii) the precipitation mixture provided in step a) comprises the metal cation precipitant in a concentration of at least 50 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM or at least 100 mM;   iii) the precipitation mixture provided in step a) comprises the metal cation precipitant in a concentration selected from 50 mM to 175 mM, 60 mM to 170 mM, 65 mM to 165 mM, 70 mM to 160 mM, 75 mM to 155 mM, 80 mM to 150 mM, 85 mM to 145 mM and 90 mM to 140 mM; and/or   iv) the precipitation buffer comprises a dissolved salt of the metal cation precipitant.   
     
     
         4 . The method according to  claim 1 , wherein the precipitation buffer comprises the metal cation precipitant in a concentration selected from 250 mM to 3 M, 500 mM to 2.8 M, 0.75 M to 2.7 M, 1 M to 2.6 M, 1.25 M to 2.5 M, 1.5 M to 2.25 M and 1.7 M to 2 M. 
     
     
         5 . The method according to  claim 1 , wherein the precipitation buffer is an aqueous solution that has a pH value selected from 2.5 to 4, 2.75 to 4.0, 2.8 to 4.0, 3.0 to 4.0, 3.1 to 3.9, 3.2 to 3.9, 3.3 to 3.9, 3.4 to 3.9, 3.5 to 3.9 and 3.5 to 3.8. 
     
     
         6 . The method according to  claim 1 , wherein the precipitation buffer is an aqueous solution that
 (i) comprises the metal cation precipitant in a concentration in the range of from 1 M to 3 M and has a pH value in the range of from 3.0 to 4.0, or   (ii) comprises the metal cation precipitant in a concentration in the range of from 1.25 M to 2.5 M and has a pH value in the range of from 3.3 to 3.9, or   (iii) comprises the metal cation precipitant in a concentration in the range of from 1.5 M to 2.25 M and has a pH value in the range of from 3.4 to 3.9, or   (iv) comprises the metal cation precipitant in a concentration in the range of from 1 M to 3 M and has a pH value in the range of from 3.5 to 3.9, or   (v) comprises the metal cation precipitant in a concentration in the range of from 1.7 M to 2.1 M and has a pH value in the range of from 3.0 to 4.0, or   (vi) comprises the metal cation precipitant in a concentration in the range of from 1.7 M to 2.1 M and has a pH value in the range of from 3.5 to 3.9.   
     
     
         7 . The method according to  claim 1 , wherein the nucleic acid to be isolated is RNA and wherein step c) comprises isolating at least small and/or large RNA from the supernatant. 
     
     
         8 . The method according to  claim 7 , wherein RNA is isolated in step c) using a nucleic acid binding solid phase and wherein at least one alcohol and/or at least one chaotropic salt is used to establish RNA binding conditions. 
     
     
         9 . The method according to  claim 1 , wherein step c) comprises:
 aa) combining at least one alcohol with the supernatant to provide a binding mixture which comprises the alcohol in a concentration of ≥35% (v/v), ≥40% (v/v) or ≥45% (v/v);   bb) binding at least large and small RNA contained in the binding mixture to a silicon containing nucleic acid binding solid phase, wherein after step bb), at least large and small RNA is bound to the solid phase;   cc) optionally washing the bound RNA; and   dd) optionally eluting RNA from the solid phase.   
     
     
         10 . The method according to  claim 1 , wherein step c) comprises combining alcohol with the supernatant to prepare binding conditions. 
     
     
         11 . The method according to  claim 9 , wherein the provided binding mixture comprises ethanol and/or isopropanol, in a concentration selected from 35% (v/v) to 70% (v/v), 40% (v/v) to 65% (v/v), 45% (v/v) to 60% (v/v) and 45% (v/v) to 55% (v/v). 
     
     
         12 . The method according to  claim 1 , wherein sample disruption occurs prior to addition of the precipitation buffer and/or at the same time/stage when the precipitation mixture is prepared. 
     
     
         13 . The method according to  claim 1 , wherein the sample is disrupted prior to step (a). 
     
     
         14 . The method according to  claim 1 , wherein the method comprises
 a) combining a precipitation buffer and at least one disruption reagent with the sample to disrupt the sample and to prepare an acidic precipitation mixture,   wherein said precipitation buffer comprises a metal cation precipitant and a buffering agent, has a pH value of 4.0 or less and does not comprise an organic solvent selected from aprotic polar solvents and protic solvents, wherein the protic solvents are linear or branched C1-C5 alcohols, and   wherein the acidic precipitation mixture comprises the metal cation precipitant in a concentration of less than 200 mM and comprises the disruption reagent and precipitating proteins;   b) separating the precipitate from the supernatant, wherein the supernatant comprises small RNA having a length of less than 200 nt and large RNA having a length of at least 1000 nt; and   c) isolating a nucleic acid from the supernatant.   
     
     
         15 . The method according to  claim 1 , having one or more of the following characteristics:
 i) total RNA is isolated from the supernatant;   ii) small RNA is isolated in form of an enriched fraction;   iii) at least mRNA is isolated from the supernatant and subsequently detected;   iv) the supernatant comprising small RNA and large RNA additionally comprises genomic DNA;   v) total nucleic acids are isolated from the supernatant;   vi) genomic DNA is isolated separately from RNA from the supernatant;   vii) extracellular nucleic acids are isolated from a cell-depleted or cell-free biological samples; and/or   vii) in step c), RNA is bound to a nucleic acid binding solid phase which optionally, is a silicon containing material such as silica, a polysilicic acid material, a borosilicate, a silicate or glass.   
     
     
         16 . The method according to  claim 1 , for isolating nucleic acids comprising or substantially consisting of RNA,
 wherein the pH value of the precipitation buffer is selected from 3.0 to 4.0, 3.1 to 3.9, 3.2 to 3.9, 3.3 to 3.9, 3.4 to 3.9, 3.5 to 3.9 and 3.5 to 3.8, and   wherein the concentration of the metal cation precipitant in the acidic precipitation mixture is selected from 50 mM to 175 mM, 60 mM to 170 mM, 65 mM to 165 mM, 70 mM to 160 mM, 75 mM to 155 mM, 80 mM to 150 mM, 85 mM to 145 mM and 90 mM to 140 mM;   and wherein said   isolating comprises isolating at least small and large RNA from the supernatant, and wherein step c) optionally comprises:
 aa) adding at least one alcohol to the supernatant to provide a binding mixture which comprises the alcohol in a concentration of ≥35% (v/v), ≥40% (v/v), ≥45% (v/v) or about 50% (v/v); 
 bb) binding nucleic acids contained in the binding mixture to a silicon containing nucleic acid binding solid phase, wherein after step bb), at least large and small RNA is bound to the solid phase; 
 cc) optionally washing the bound RNA; 
 dd) eluting RNA from the solid phase. 
   
     
     
         17 . The method according to  claim 1 , wherein the precipitation buffer is added to the sample in a ratio in the range selected from 1:1 to 1:30, 1:5 to 1:20, 1:8 to 1:17, 1:10 to 1:15, and 1:12 to 1:14 (precipitation buffer:sample). 
     
     
         18 . A precipitation buffer for precipitating proteins comprising at least one metal cation precipitant and at least one buffering agent, wherein the precipitation buffer has a pH value of 4 or less and wherein the precipitation buffer does not comprise an organic solvent selected from aprotic polar solvents and protic solvents, wherein the protic solvents are linear or branched C1-C5 alcohols. 
     
     
         19 . (canceled) 
     
     
         20 . The precipitation buffer according to  claim 18 , having one or more of the following characteristics:
 i) the metal cation precipitant is selected from Zn 2+ , Cu 2+  and Al 3+ ;   ii) the buffering agent is or is derived from a carboxylic acid or phosphate;   iii) it comprises the metal cation precipitant in a concentration selected from 250 mM to 3 M, 500 mM to 2.8 M, 0.75 M to 2.7 M, 1 M to 2.6 M, 1.25 M to 2.5 M, 1.5 M to 2.25 M and 1.7 M to 2 M;   iv) it has a pH value that lies in a range selected from 2.5 to 4, 2.75 to 4.0, 2.8 to 4.0, 3.0 to 4.0, 3.1 to 3.9, 3.2 to 3.9, 3.3 to 3.9, 3.4 to 3.9, 3.5 to 3.9 and 3.5 to 3.8;   v) it comprises the buffering agent in a concentration selected from 300 mM to 3 M, 600 mM to 2.75 M, 900 mM to 2.5 M, 1.2 M to 2.4 M, 1.4 M to 2.3 M and 1.5 M to 2.25 M;   vi) it comprises the metal cation precipitant in form of a dissolved salt;   and/or   vi) the precipitation buffer
 aa) comprises Zn 2+ , Cu 2+  or Al 3+  as metal cation precipitant in form of a dissolved salt in a concentration selected from 250 mM to 3 M, 500 mM to 2.8 M, 0.75 M to 2.7 M, 1 M to 2.6 M, 1.2 M to 2.5 M, 1.3 M to 2.25 M and 1.6 M to 2 M; and 
 bb) has a pH value that lies in a range selected from 3.3 to 3.9, 3.4 to 3.9, 3.5 to 3.9 and 3.5 to 3.8. 
   
     
     
         21 . (canceled) 
     
     
         22 . The method according to  claim 1 , wherein the metal cation precipitant is Zn 2+ . 
     
     
         23 . The method according to  claim 10 , wherein the alcohol is selected from isopropanol and ethanol. 
     
     
         24 . The method according to  claim 16 , wherein the at least one alcohol in step aa) is isopropanol.