METHOD FOR ISOLATION OF NUCLEIC ACID CONTAINING PARTICLES AND EXTRACTION OF NUCLEIC ACIDS THEREFROM
A method for extracting nucleic acids from a biological sample by isolating nucleic acid-containing particles from the biological sample by one or more centrifugation procedures, performing one or more steps to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction, and extracting nucleic acids from the isolated particles. The centrifugation procedures are performed at a speed not exceeding about 200,000 g. The extracted nucleic acids contain both 18S and 28S rRNA.
1 - 16 . (canceled)
17 . A method for extracting nucleic acids from a biological sample from a subject, comprising the steps of:
a) centrifuging the biological sample with one or more centrifugation procedures to generate isolated, pelleted exosomes, wherein none of the centrifugation procedures are performed at a speed exceeding 20,000 g; and
b) extracting nucleic acids from the isolated, pelleted exosomes, wherein the extracted nucleic acids comprise RNA molecules having a length of less than 200 nucleotides.
18 . The method of claim 17 , wherein the extracted nucleic acids comprise RNA, DNA, or both RNA and DNA.
19 . The method of claim 17 , wherein the extracted nucleic acids comprise 18S and 28S rRNAs.
20 . The method of claim 17 , wherein the method further comprises analyzing the extracted nucleic acids.
21 . The method of claim 20 , wherein analyzing the extracted nucleic acids comprises determining the presence or absence of a biomarker in the extracted nucleic acids.
22 . The method of claim 20 , wherein analyzing the extracted nucleic acids includes analysis of one or more of: expression levels, alternative splicing variants, chromosome rearrangements, gene copy numbers, nucleic acid modifications, single nucleotide polymorphisms, and mutations for one or more of the extracted nucleic acids.
23 . The method of claim 20 , wherein analyzing the extracted nucleic acid comprises nucleic acid methylation analysis of one or more of the extracted nucleic acids.
24 . The method of claim 17 , wherein the method further comprises treating the biological sample and/or the isolated, pelleted exosomes with an RNase inhibitor, a DNase, or an RNase inhibitor and a DNase.
25 . The method of claim 17 , wherein the biological sample is a urine sample, a serum sample, or a spinal fluid sample from the subject.
26 . The method of claim 17 , wherein the subject is a human.
27 . A method for extracting nucleic acids from a biological sample from a subject, comprising the steps of:
a) centrifuging the biological sample with one or more centrifugation procedures to generate isolated, pelleted exosomes, wherein none of the centrifugation procedures are performed at a speed exceeding 20,000 g;
b) extracting nucleic acids from the isolated, pelleted exosomes; and
c) analyzing the extracted nucleic acids.
28 . The method of claim 27 , wherein analyzing the extracted nucleic acids comprises determining the presence or absence of a biomarker in the extracted nucleic acids.
29 . The method of claim 27 , wherein analyzing the extracted nucleic acids includes analysis of one or more of: expression levels, alternative splicing variants, chromosome rearrangements, gene copy numbers, nucleic acid modifications, single nucleotide polymorphisms, and mutations for one or more of the extracted nucleic acids.
30 . The method of claim 27 , wherein analyzing the extracted nucleic acid comprises nucleic acid methylation analysis of one or more of the extracted nucleic acids.
31 . The method of claim 27 , wherein the extracted nucleic acids comprise RNA, DNA, or both RNA and DNA.
32 . The method of claim 27 , wherein the extracted nucleic acids comprise 18S and 28S rRNAs.
33 . The method of claim 27 , wherein the method further comprises treating the biological sample and/or the isolated, pelleted exosomes with an RNase inhibitor, a DNase, or an RNase inhibitor and a DNase.
34 . The method of claim 27 , wherein the biological sample is a urine sample, a serum sample, or a spinal fluid sample from the subject.
35 . The method of claim 27 , wherein the subject is a human.