US 12,319,909 B2
Compositions and methods for ribonucleic acid extraction
Yin To Chiu, Hong Kong (CN); Harsha Madan Kittur, Hong Kong (CN); Cheuk Yiu Tenny Chung, Hong Kong (CN); and Garrett Lee Mosley, Hong Kong (CN)
Assigned to Phase Scientific International, Ltd., Hong Kong (CN)
Filed by Phase Scientific International, Ltd., Hong Kong (CN)
Filed on Mar. 19, 2021, as Appl. No. 17/249,936.
Claims priority of provisional application 62/992,222, filed on Mar. 20, 2020.
Prior Publication US 2021/0292743 A1, Sep. 23, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1003 (2013.01) 24 Claims
 
1. A method for isolating ribonucleic acids (RNAs) from a fluid mixture containing RNAs and proteins, wherein the method comprises:
(a) providing a biological sample;
(b) mixing the biological sample with a lysing reagent to form a sample mixture, wherein the lysing reagent has a concentration of 2M-20M, and the lysing reagent is a lysing salt or a detergent, wherein
the lysing salt is selected from the group consisting of NaCl, KCl, (NH4)2SO4, guanidinium hydrochloride and ammonium chloride; and
the detergent is selected from the group consisting of Triton X-100, Nonidet P-40, sodium deoxycholate, sodium dodecyl sulfate (SDS), 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), and ethyl trimethyl ammonium bromide;
(c) incubating the sample mixture to arrive at the fluid mixture;
(d) adding said fluid mixture to an aqueous two-phase system (ATPS) comprising a first phase solution and a second phase solution, wherein the first phase solution comprises 10-25% w/w polymer and the second phase solution comprises 50-70% w/w salt, wherein the polymer has a molecular weight of less than 2,000 Da and is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), copolymer mixture of ethylene oxide and propylene oxide (EOPO), polyacrylate, and 3-(2-methylpropoxy)propan-1-ol; and the salt is selected from the group consisting of potassium phosphate, sodium citrate, sodium phosphate, ammonium sulfate, and sodium sulfate; such that the RNAs partition to the first phase solution and said proteins partition to the second phase solution; and
(e) isolating the RNAs from the first phase solution.