Isolating circulating microRNA (miRNA)
Methods for isolating circulating small RNAs, e.g., microRNA (miRNA), from plasma samples, e.g., that comprise using an alkaline phenol:chloroform extraction, and methods of use thereof, including for the detection, prognosis, and/or monitoring of disease in a subject.
1. A method for isolating RNA from a sample, the method comprising:
digesting the sample with Proteinase K in the presence of chaotropic salts and detergent;
extracting RNA from the sample using an alkaline phenol:chloroform extraction; and
isolating the extracted RNA from the sample.
2. The method of claim 1 , wherein the phenol:chloroform is at pH 8.0.
3. The method of claim 1 , wherein the sample is a sample of a biological fluid.
4. The method of claim 2 , wherein the biological fluid comprises blood, plasma or serum, urine, cerebral spinal fluid (CSF) or other fluid.
5. The method of claim 1 , wherein extracting RNA from the sample using alkaline phenol:chloroform extraction comprises contacting the sample with a solution comprising nuclease free water and phenol:chloroform.
6. The method of claim 4 , wherein the nuclease free water and phenol:chloroform are at a ratio of at least 1:10 (vol:vol) of nuclease free water to phenol:chloroform.
7. The method of claim 4 , wherein the nuclease free water and phenol:chloroform are at a ratio of at least an equal volume of nuclease free water and phenol:chloroform.
8. The method of claim 1 , wherein the phenol:chloroform ratio is between 6:1 to 1:1.
9. The method of claim 1 , wherein the phenol:chloroform ratio is between 5:1 to 3:1.
10. The method of claim 1 , wherein the phenol:chloroform ratio is 5:1.
11. The method of claim 1 , wherein lysing the sample comprises contacting the sample with a detergent buffer in the presence of Proteinase K.
12. The method of claim 10 , comprising contacting the sample with at least 6 mAU of Proteinase K.
13. The method of claim 10 , wherein the detergent buffer is a guanidine buffer.
14. The method of claim 5 , wherein the guanidine buffer comprises Guanidine HCl 2M; Triton 1.6% (vol/vol); Tween 20 1.6% (vol/vol); EDTA 37.5 mM; and Tris pH 8.0 37.5 mM.
15. The method of claim 1 , wherein isolating the extracted RNA from the sample comprises one or more of ethanol extraction; magnetic bead separation; or spin column extraction.
16. A method of detecting a level of a circulating miRNA in a subject, the method comprising:
providing a sample comprising plasma or serum from a subject;
lysing the sample;
extracting RNA from the sample using ph 8.0 phenol:chloroform extraction;
isolating the extracted RNA from the sample;
and determining a level of the miRNA in the extracted RNA.
17. The method of claim 16 , wherein determining a level of the miRNA in the extracted RNA comprises amplifying the miRNA; sequencing the RNA; hybridization; or a gene chip.
18. A method of monitoring a level of a miRNA in a subject, the method comprising:
providing a first sample comprising plasma or serum of the human subject at a first time point;
detecting a level of one or more circulating miRNAs in the first sample using the method of claim 1 ;
providing a second sample comprising plasma or serum of the human subject at a second time point;
detecting a level of the one or more circulating miRNAs in the second sample using the method of claim 1 ; and
comparing the level of the miRNA in the first sample to the level of the miRNA in the second sample.