# US 10,767,215 B2 · Stabilizing compositions and methods for extraction of ribonucleic acid

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- Number: US 10,767,215 B2
- Document ID: US10767215B2
- Status: Granted
- Kind: B2
- Application number: 15/980,682
- Filed: 2018-05-15
- Published: 2018-12-20
- Granted: 2020-09-08
- Earliest claimed: 2006-10-06
- Assignees: Dna Genotek Inc.
- Applicants: DNA Genotek Inc.
- Inventors: Hyman Chaim Birnboim; Adele Jackson
- Web page: https://patentgrep.com/patent/US10767215B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## 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.

## Claims

### Claim 1

A phenol-free method for preserving ribonucleic acid from a biological sample comprising the steps of:
a. obtaining the sample from a subject;
b. contacting the sample with a composition comprising an anionic detergent, wherein the anionic detergent is at a concentration of 1% to 8%, and a buffering agent at a pH of about 5 to about 8.2 to form a mixture; and
c. storing the mixture at room temperature;
wherein the composition stabilizes the ribonucleic acid at room temperature.

### Claim 2 (depends on 1)

The method of claim 1, further comprising contacting the mixture with a protease following step c.

### Claim 3 (depends on 2)

The method of claim 2, wherein the protease is proteinase K.

### Claim 4 (depends on 1)

The method of claim 1 further comprising heating the mixture following step c, wherein the mixture is heated at a temperature of greater than or equal to about 50° C.

### Claim 5 (depends on 4)

The method of claim 4, wherein the mixture is heated at a temperature of greater than or equal to about 90° C.

### Claim 6 (depends on 1)

The method of claim 1, wherein the anionic detergent is sodium dodecyl sulphate, sodium lauroyl sarcosinate, lithium dodecyl sulphate or sodium 1-octane sulfonic acid.

### Claim 7 (depends on 1)

The method of claim 1, wherein the anionic detergent is sodium dodecyl sulphate or sodium sarcosinate.

### Claim 8 (depends on 1)

The method of claim 1, wherein the buffering agent is at a pH of about 5.1 to about 7.

### Claim 9 (depends on 1)

The method of claim 1, wherein the 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), or combinations thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the buffering agent is a phosphate buffer, a carbonate buffer, an ethylenediamine buffer or an imidazole buffer.

### Claim 11 (depends on 1)

The method of claim 1, wherein the buffering agent is CDTA or citric acid.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sample is a bodily fluid or a bodily tissue from a mammal.

### Claim 13 (depends on 12)

The method of claim 12, wherein the mammal is a human or a cow.

### Claim 14 (depends on 1)

The method of claim 1, wherein the 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.

### Claim 15 (depends on 1)

The method of claim 1, wherein the composition stabilizes the ribonucleic acid at room temperature for one or more of the following time periods: 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.

### Claim 16 (depends on 15)

The method of claim 15, wherein the composition stabilizes the ribonucleic acid at room temperature for about one day to about eight weeks at room temperature.

### Claim 17 (depends on 1)

The method of claim 1, wherein the buffering agent is at a pH of about 5.5 to about 7.5.

### Claim 18 (depends on 1)

The method of claim 1, wherein the buffering agent is at a pH of about 6.5 to about 7.0.

### Claim 19 (depends on 1)

The method of claim 1, wherein the buffering agent is at a pH of about 6.8.