Stabilizing compositions and methods for extraction of ribonucleic acid

Abstract

The present invention provides a composition and method for stabilizing ribonucleic acid (RNA) from biological samples such that the ribonucleic acid within the sample remains stable at room temperature. The composition comprises an anionic detergent and a buffering agent at a pH of about 5 to about 8.2 and is used in methods for extracting and storing ribonucleic acid from the biological sample.

1 . A composition for extracting and storing ribonucleic acid from a sample such that the ribonucleic acid within said sample remains stable at room temperature, said composition comprises:
 a. an anionic detergent; and   b. a buffering agent at a pH of about 5 to about 8.2;   wherein when said composition is mixed with said sample to form a liquid mixture, the ribonucleic acid in said liquid mixture is stable at room temperature.   
   
   
       2 . The composition of  claim 1 , wherein said anionic detergent is sodium dodecyl sulphate, sodium lauroyl sarcosinate (Sarkosyl), lithium dodecyl sulphate or sodium 1-octane sulfonic acid. 
   
   
       3 . The composition of  claim 1  or  2 , wherein said anionic detergent is sodium dodecyl sulphate or sodium lauroyl sarcosinate. 
   
   
       4 . The composition of  claim 3 , wherein the sodium dodecyl sulphate or sodium lauroyl sarcosinate are at a concentration of from about 0.5% to about 8% when mixed with said sample. 
   
   
       5 . The composition of any one of  claims 1  to  4 , wherein said buffering agent is at a pH of about 5.1 to about 7, about 5.5 to about 7.5, about 6.5 to about 7.0 or about 6.8. 
   
   
       6 . The composition of any one of  claims 1  to  5 , wherein said buffering agent is sodium cyclohexane diaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic acid or acetate (e.g. sodium acetate), citric acid or citrate, malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric acid, maleic acid, cacodylic acid, ββ′-Dimethylglutaric acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole, glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate, 2:4:6 collidine, 5(4)-methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid, tris(hydroxymethyl)aminomethane (Tris), 3-(N-Morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), analogues thereof, or combinations thereof. 
   
   
       7 . The composition of any one of  claims 1  to  5 , wherein said buffer is a phosphate buffer, a carbonate buffer, an ethylenediamine buffer or an imidazole buffer. 
   
   
       8 . The composition of any one of  claims 1  to  6 , wherein the buffering agent is CDTA or citric acid. 
   
   
       9 . The composition of  claim 1 , wherein said sample is a bodily fluid or a bodily tissue from a mammal. 
   
   
       10 . The composition of  claim 9 , wherein said mammal is a human or a cow. 
   
   
       11 . The composition of  claim 1 , wherein said sample is from a human; a non-human primate; livestock including cattle, pigs, sheep, goats or domestic birds including chicken, turkey, pheasant, duck or geese); game or wild animals including deer, elk, moose, fish, birds or bears; laboratory or companion animals including non-human primates, rodents including mice, rats, rabbits, guinea pigs, gerbils or hamsters; dogs; cats; fish; snakes; lizards; turtles; a horse; plants; plant parts; cell lines; soil microorganisms; sewage microorganisms; or pathogenic microorganisms including virus, bacteria or parasites. 
   
   
       12 . The composition of any one of  claims 1  to  11 , wherein the composition stabilized said ribonucleic acid at room temperature for at least about one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks or eight weeks, about one day to about eight weeks, or greater than about eight weeks. 
   
   
       13 . The composition of any one of  claims 1  to  12 , wherein the composition stabilizes said ribonucleic acid at room temperature for about one day to about eight weeks at room temperature. 
   
   
       14 . A method for preserving ribonucleic acid from a biological sample comprising the steps of:
 a. obtaining the sample from a subject;   b. contacting said sample with a composition comprising an anionic detergent and a buffering agent at a pH of about 5 to about 8.2 to form a liquid mixture;   c. storing the mixture at room temperature; and   d. heating the mixture at greater than or about equal to 50° C. prior to subsequent processing,   
     wherein said composition stabilizes said ribonucleic acid at room temperature. 
   
   
       15 . The method of  claim 14 , wherein step d further comprises contacting the mixture with a protease. 
   
   
       16 . The method of  claim 15 , wherein the protease is proteinase K. 
   
   
       17 . The method of any one of  claims 14  to  16  further comprising the step of (e) heating the mixture at greater than or equal to about 90° C. prior to subsequent processing and before or after heating step d. 
   
   
       18 . The method of anyone of  claims 14  to  17 , wherein said anionic detergent is sodium dodecyl sulphate, sodium lauroyl sarcosinate, lithium dodecyl sulphate or sodium 1-octane sulfonic acid. 
   
   
       19 . The method of any one of  claims 14  to  18 , wherein said anionic detergent is sodium dodecyl sulphate or sodium lauroyl sarcosinate. 
   
   
       20 . The method of  claim 19 , wherein the sodium dodecyl sulphate or sarkoryl are at a concentration of from about 0.5% to about 8%. 
   
   
       21 . The method of any one of  claims 14  to  20 , wherein said buffering agent is at a pH of about 5.1 to about 7, about 5.5 to about 7.5, about 6.5 to about 7.0 or about 6.8. 
   
   
       22 . The method of any one of  claims 14  to  21 , wherein said buffering agent is sodium cyclohexane diaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic acid or acetate (e.g. sodium acetate), citric acid or citrate, malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric acid, maleic acid, cacodylic acid, ββ′-Dimethylglutaric acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole, glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate, 2:4:6-collidine, 5(4)-methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid, tris(hydroxymethyl)aminomethane (Tris), 3-(N-Morpholino)propanesulfonic acid; 4-morpholinepropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-[tris(hydroxymethypmethyl]-2-aminoethanesulfonic acid (TES), 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), or combinations thereof. 
   
   
       23 . The method of any one of  claims 14  to  22 , wherein said buffering agent is a phosphate buffer, a carbonate buffer, an ethylenediamine buffer or an imidazole buffer. 
   
   
       24 . The method of any one of  claims 14  to  22 , wherein the buffering agent is CDTA or citric acid. 
   
   
       25 . The method of any one of  claims 14  to  24 , wherein said sample is a bodily fluid or a bodily tissue from a mammal. 
   
   
       26 . The method of  claims 25 , wherein said mammal is a human or a cow. 
   
   
       27 . The method of  claim 14 , wherein said sample is from a human; a non-human primate; livestock including cattle, pigs, sheep, goats or domestic birds including chicken, turkey, pheasant, duck or geese); game or wild animals including deer, elk, moose, fish, birds or bears; laboratory or companion animals including non-human primates, rodents including mice, rats, rabbits, guinea pigs, gerbils or hamsters; dogs; cats; fish; snakes; lizards; turtles; a horse; plants; plant parts; cell lines; soil microorganisms; sewage microorganisms; or pathogenic microorganisms including virus, bacteria or parasites. 
   
   
       28 . The method of any one of  claims 14  to  27 , wherein the composition stabilized said ribonucleic acid at room temperature for at least about one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks or eight weeks, about one day to about eight weeks, or greater than about eight weeks. 
   
   
       29 . The method of any one of  claims 14  to  27 , wherein the composition stabilizes said ribonucleic acid at room temperature for about one day to about eight weeks at room temperature. 
   
   
       30 . A RNA storage kit, comprising:
 a. a composition according to any one of  claims 1  to  13 ; and   b. instructions for the use thereof.