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. A method for extracting nucleic acids from a biological sample from a subject, comprising the steps of:
a. isolating microvesicles comprising exosomes from the biological sample by one or more centrifugation procedures to pellet the microvesicles from the biological sample,
wherein none of the centrifugation procedures are performed at a speed exceeding about 20,000 g, and
wherein none of the centrifugation procedures use nanomembrane ultrafiltration concentration; and
b. extracting nucleic acids from the isolated, pelleted microvesicles comprising exosomes,
wherein 18S and 28S rRNAs are detectable in the extracted nucleic acids, and
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.
2. The method of claim 1 , wherein the biological sample is a body fluid.
3. The method of claim 2 , wherein the body fluid is a serum, urine, or spinal fluid sample from the subject.
4. The method of claim 3 , wherein the subject is a human or other mammal.
5. The method of claim 1 , wherein the extracted nucleic acids comprise RNA, DNA, or both RNA and DNA.
6. 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.
7. The method of claim 1 , 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.
8. The method of claim 1 , further comprising treating the biological sample and/or the isolated microvesicles with DNase, RNase inhibitor, or DNase and RNase inhibitor.
9. The method of claim 1 , further comprising treating the biological sample with RNase inhibitor before isolating the microvesicles.