US2013295574A1PendingUtilityA1

Method for Isolation of Nucleic Acid Containing Particles and Extraction of Nucleic Acids Therefrom

Individually held — no corporate assignee on recordPriority: Nov 10, 2010Filed: Nov 10, 2011Published: Nov 7, 2013

Est. expiryNov 10, 2030(~4.3 yrs left)· nominal 20-yr term from priority

Inventors:Johan Karl Olov Skog Leileata M. Russo

C12N 15/1003B01D 21/26B01D 21/262

PatentIndex Score

Cited by

References

Claims

Abstract

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.

Claims

exact text as granted — not AI-modified

1 . A method for extracting nucleic acids from a biological sample, comprising the steps of:
 a. isolating nucleic acid-containing particles from the biological sample by one or more centrifugation procedures, wherein none of the centrifugation procedures are performed at a speed exceeding about 200,000 g;   b. performing one or more steps to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction; and   c. extracting nucleic acids from the isolated particles.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid-containing particles are microvesicles, RNA-protein complexes, DNA-protein complexes, or a combination of any of microvesicles, RNA-protein complexes, and DNA-protein complexes. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid-containing particles are microvesicles. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid-containing particles are RNA-protein complexes. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid-containing particles are DNA-protein complexes. 
     
     
         6 . The method of  claim 1 , wherein all of the centrifugation procedures are performed at speeds of about 2,000 g to about 200,000 g. 
     
     
         7 . The method of  claim 1 , wherein none of the centrifugation procedures are performed at a speed exceeding about 50,000 g. 
     
     
         8 . The method of  claim 1 , wherein none of the centrifugation procedures are performed at a speed exceeding about 20,000 g. 
     
     
         9 . The method of  claim 1 , wherein the biological sample is a body fluid. 
     
     
         10 . The method of  claim 9 , wherein the body fluid is a serum, urine, or spinal fluid sample from a subject. 
     
     
         11 . The method of  claim 10 , wherein the subject is a human or other mammal. 
     
     
         12 . The method of  claim 1 , wherein the extracted nucleic acids comprise RNA, DNA, or both RNA and DNA. 
     
     
         13 . The method of  claim 1 , wherein the extracted nucleic acids comprise one or more nucleic acids having a sequence more than 90% homologous to the nucleic acid sequence corresponding to any of the genes consisting of EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4, CD40, CD4OLG, CDS, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CD8B, CD96, CHST10, COLIA1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS, FCER1A, FCER2, FCGR1A/FCGR1B/FCGR1C, HLA-A/HLA-A29.1, HLA-DRA, ICAM2, IL 12RB1, IL1R2, IL2RA, ITGA1, ITGA2, ITGA3, KLRB1, KLRC1, KLRD1, KRT18, KRT5, KRT8/LOC728638, MS4A1, MYH10, MYH9, MYOCD, NCAM1, NOS3, NT5E, PECAM1, RETN, S100A8, SELP, ST6GAL1, EPCAM, TEK, TNFRSF4, TNFRSF8, TPSAB1/TPSB2, VCAM1, or VWF. 
     
     
         14 . The method of  claim 1 , wherein 18S and 28S rRNAs are detectable in the extracted nucleic acids. 
     
     
         15 . The method of  claim 13 , wherein the ratio of the amount of 18S rRNA to the amount of 28S rRNA, as detected in the extracted nucleic acids, is about 0.5 to about 1.0. 
     
     
         16 . The method of  claim 13 , wherein the ratio of the amount of 18S rRNA to the amount of 28S rRNA, as detected in the extracted nucleic acids, is about 0.5. 
     
     
         17 . The method of  claim 1 , wherein step (b) is achieved by treating the biological sample and/or the isolated particles with DNase, RNase inhibitor, or DNase and RNase inhibitor. 
     
     
         18 . The method of  claim 1 , wherein step (b) comprises a step of treating the biological sample with RNase inhibitor before isolating the particles. 
     
     
         19 . A nucleic acid sample obtained by the method of  claim 1 . 
     
     
         20 . The use of the method of  claim 1  for aiding in the diagnosis of a subject, wherein the presence or absence of a biomarker within the extracted nucleic acids is determined, and said biomarker is associated with a disease or other medical condition in the subject. 
     
     
         21 . The use of the method of  claim 1  for monitoring the progress or reoccurrence of a disease or other medical condition in a subject, wherein the presence or absence of a biomarker within the extracted nucleic acids is determined, and said biomarker is associated with the progress or reoccurrence of a disease or other medical condition in the subject. 
     
     
         22 . The use of the method of  claim 1  for aiding in the evaluation of treatment efficacy for a subject undergoing or contemplating treatment for a disease or other medical condition, wherein the presence or absence of a biomarker within the extracted nucleic acids is determined, and said biomarker is associated with treatment efficacy for the subject undergoing or contemplating treatment for a disease or other medical condition. 
     
     
         23 . The use of any of  claims 20 - 22   claim 20 , wherein the biomarker is a nucleic acid corresponding to any one or more of the genes consisting of EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4, CD40, CD4OLG, CDS, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CD8B, CD96, CHST10, COL1A1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS, FCER1A, FCER2, FCGR1A/FCGR1B/FCGR1C, HLA-A/HLA-A29.1, HLA-DRA, ICAM2, IL 12RB1, IL1R2, IL2RA, ITGA1, ITGA2, ITGA3, KLRB1, KLRC1, KLRD1, KRT18, KRT5, KRT8/LOC728638, MS4A1, MYH10, MYH9, MYOCD, NCAM1, NOS3, NT5E, PECAM1, RETN, S100A8, SELP, ST6GAL1, EPCAM, TEK, TNFRSF4, TNFRSF8, TPSAB1/TPSB2, VCAM1, or VWF. 
     
     
         24 . A kit for use in the method of  claim 1 , comprising the following components:
 (a) RNase inhibitor in a quantity sufficient to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction;   (b) RNA purification reagent;   (c) optionally, lysis buffer;   (d) optionally, DNase; and   (e) optionally, instructions for using the foregoing reagents in the extraction of nucleic acids from isolated particles.

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