Method of single strand RNA purification employing an anion exchanger
A method of single strand RNA purification employing an anion exchanger comprising the steps applying a sample containing single-stranded RNA to an anion exchanger, washing the anion exchanger at a first temperature in the range of 18° C. to 25° C. with a first salt solution having a first ionic strength in the range 0.5 M to 12.0 M, eluting the single stranded RNA by a second salt solution having a second ionic strength at a second temperature in the range of 35° C. to 80° C., with the proviso that the first ionic strength is at least 0.5 M higher than the second ionic strength.
1 . A method of single strand RNA purification employing an anion exchanger comprising the steps of:
applying a sample containing single-stranded RNA to an anion exchanger,
washing the anion exchanger at a first temperature in the range of 18° C. to 25° C. with a first salt solution having a first ionic strength in the range of 0.5 M to 12.0 M,
eluting the single stranded RNA by a second salt solution having a second ionic strength at a second temperature in the range of 35° C. to 80° C.,
with the proviso that the first ionic strength is at least 0.5 M higher than the second ionic strength.
2 . The method claim 1 wherein the washing with the first salt solution is conducted at an ionic strength in the range of 1 M to 10 M.
3 . The method of claim 1 wherein the washing with the first salt solution is conducted with a chaotropic salt.
4 . The method of claim 3 wherein the chaotropic salt is selected from the group consisting of a guanidine salt, a perchlorate salt, a thiocyanate salt, and mixtures thereof.
5 . The method of claim 1 wherein the washing with the first salt solution includes a chelating agent at a concentration in the range of 1 mM to 1000 mM.
6 . The method of claim 1 wherein the washing with the first salt solution includes one or more species of chelating agents selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and tris(2-aminoethyl)amine (TREN).
7 . The method of claim 1 wherein the second salt solution employed for eluting the single-stranded RNA is applied at the second temperature in the range of 45° C. to 70° C.
8 . The method of claim 1 wherein the anion exchanger is selected from the group consisting of a strong anion exchanger bearing quaternary amino ion exchange groups, a weak anion exchanger bearing tertiary amino ion exchange groups, a weak anion exchanger bearing secondary amino ion exchange groups, a weak anion exchanger bearing primary amino groups and an anion exchanger bearing more than one kind of amino groups.
9 . The method of claim 1 wherein the size of the RNA is in the range of 1,000 bases to 25,000 bases.
10 . The method claim 1 wherein the washing with the first salt solution is conducted at an ionic strength in the range of 2 M to 8 M.
11 . The method claim 1 wherein the washing with the first salt solution is conducted at an ionic strength in the range of 4 M to 6 M.
12 . The method of claim 1 wherein the washing with the first salt solution includes a chelating agent at a concentration in the range of 5 mM to 500 mM.
13 . The method of claim 1 wherein the washing with the first salt solution includes a chelating agent at a concentration in the range of 10 mM to 100 mM.
14 . The method of claim 1 wherein the washing with the first salt solution includes a chelating agent at a concentration in the range of 20 mM to 50 mM.
15 . The method of claim 1 wherein the second salt solution employed for eluting the single-stranded RNA is applied at the second temperature in the range of 50° C. to 70° C.
16 . The method of claim 1 wherein the second salt solution employed for eluting the single-stranded RNA is applied at the second temperature in the range of 55° C. to 70° C.
17 . The method of claim 1 wherein the second salt solution employed for eluting the single-stranded RNA is applied at the second temperature in the range of 60° C. to 70° C.