Identification of microRNAs (miRNAs) in fecal samples as biomarkers for gastroenterological cancers
A simple, rapid, inexpensive, and promising commercial biomarker assay method for multiple diseases is described herein. The present invention detects miRNA-based biomarkers in human stool specimens. The method of the present invention amplifies miRNA directly from stool specimens without any prior miRNA extraction. Differential expression of specific microRNAs in stool of colorectal cancer CRC and adenoma patients suggest fecal microRNAs as a novel potential biomarker for colorectal neoplasia detection. The method of the present invention has diagnostic, prognostic, and therapeutic relevance for gastroenterological cancers/colorectal cancer and as well as further acquired or hereditary GI diseases.
1. A method of processing a biological sample comprising ribonucleic acids of a subject for characterization of one or more target MicroRNAs (miRNAs) in the sample comprising the steps of:
a) mixing the sample with RNase free water, a salt solution, or both to form a suspension;
b) centrifuging the suspension;
c) separating an miRNA-containing supernatant from the centrifuged suspension;
d) amplifying the one or more miRNAs without prior RNA extraction;
wherein the sample containing ribonucleic acid is mixed with RNase free water, a salt solution, or both without a prior extraction.
2. The method of claim 1 , wherein the biological sample contains miRNA in exosomes.
3. The method of claim 1 , wherein the biological sample is a stool sample.
4. The method of claim 1 , wherein the salt is an ionic salt selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, iron salts, and quarternary ammonium salts.
5. The method of claim 1 , wherein a ratio of the biological sample to the salt solution is selected from the group consisting of 1:1, 1:2, 1:5, 1:10, 1:20, 1:25, 1:50, and 1:100.
6. The method of claim 5 , wherein the salt solution is a sodium chloride solution.
7. The method of claim 1 , further comprising storing the supernatant at −80.degree. C. following step c).
8. The method of claim 1 , further comprising measuring a total RNA/microRNA (miRNA) concentration in the supernatant using a spectrophotometer before amplification.
9. The method of claim 1 , wherein the amplifying of the miRNA comprises transcribing a cDNA from the RNA-containing supernatant using one or more specific miRNA primers; amplifying the transcribed cDNA using a polymerase chain reaction assay to obtain one or more amplified miRNAs.
10. The method of claim 9 , wherein the specific miRNA primers are specific for miR-21.
11. The method of claim 9 , wherein the specific miRNA primers are specific for miR-106a.
12. The method of claim 9 , wherein said characterization of one or more target miRNAs aids in detecting a disease in a subject suspected of having the disease, said method further comprising comparing the levels of the one or more miRNAs from the sample containing ribonucleic acid of the subject suspected of having the disease with that of one or more healthy subjects, and wherein an elevated level of the one or more miRNAs indicates the presence of the disease.
13. The method of claim 12 , wherein the disease is a cancer.
14. The method of claim 12 , wherein the disease is a gastroenterological disease.
15. The method of claim 12 , wherein the disease is colorectal or gastroenterological cancer.
16. The method of claim 12 , wherein the disease is colon adenoma, colon adenocarcinoma, or colorectal cancer.
17. The method of claim 12 , wherein endoscopic examination of the subject is carried out if and only if the one or more miRNAs indicating presence of the disease is elevated.
18. The method of claim 12 , wherein if the one or more miRNAs indicating the presence of the disease is elevated, then the method further comprises endoscopic removal of polyps and/or adenomas from the subject.
19. The method of claim 12 , wherein the target miRNA is miR-21.
20. The method of claim 15 , wherein the target miRNA is miR-21.
21. The method of claim 1 , wherein the biological sample is a blood sample.