Methods for identifying medical conditions in human subjects

Abstract

This invention provides in vitro and non-invasive methods for detecting medical conditions in subjects. [Solution] The method includes: concentrating very small embryoid stem cells from a sample to obtain a mixture containing the very small embryoid stem cells; obtaining nucleic acids from the mixture of steps; performing an assay on the nucleic acids to analyze the expression level of Oct4A in the very small embryoid stem cells from the sample; and comparing the expression level of Oct4A in the very small embryoid stem cells from the sample with the expression level of Oct4A in a control sample. This disclosure also provides a method for predicting the onset of cancer and predicting the presence of cancer. A method for treating cancer is also disclosed herein. Furthermore, reagent kits and detection kits are also disclosed.

Claims

1. An in vitro method for assisting in the detection of the absence of cancer in a subject, wherein the method is (a) Concentrating very small embryoid stem cells from the whole blood sample of the subject to obtain a mixture containing the very small embryoid stem cells, (b) Obtaining nucleic acids from the mixture, (c) Performing an assay on the nucleic acid to analyze the expression level of Oct4A in the very small embryoid stem cells from the whole blood sample, (d) Comparing the expression level of Oct4A in the very small embryoid stem cells from the whole blood sample with the reference expression level of Oct4A in a non-cancer subject, Compared to the expression level of Oct4A in subjects without cancer, it is detected whether the expression level of Oct4A in the very small embryoid stem cells from the whole blood sample is less than a 1.1-fold increase. The method described above is an in vitro method that does not rely on invasive techniques.

2. The method according to claim 1, further comprising analyzing the nucleic acid by performing a sequence-based assay.

3. The method according to claim 1 or 2, wherein the nucleic acid is obtained from the mixture by a method selected from the group consisting of (i) guanidinium thiocyanate-phenol-chloroform nucleic acid extraction, (ii) cesium chloride gradient centrifugation, (iii) cetyltrimethylammonium bromide nucleic acid extraction, (iv) alkaline extraction, (v) resin-based extraction, and (vi) solid-phase nucleic acid extraction.

4. The method according to any one of claims 1 to 3, wherein the assay is performed on the nucleic acid to analyze the expression level of Oct4A by a technique selected from the group consisting of quantitative polymerase chain reaction (PCR), flow cytometry, and next-generation sequencing (NGS).

5. The concentration of the very small embryoid stem cells from the whole blood sample is i) To obtain a first mixture by contacting the whole blood sample with a neutral buffer in a ratio in the range of 1:1 to 1:20, ii) The first mixture is centrifuged to obtain a second mixture containing a red blood cell (RBC) fraction, iii) The method according to any one of claims 1 to 4, comprising processing the second mixture to obtain concentrated very small embryoid stem cells.

6. The method according to claim 5, wherein the processing of the second mixture comprises (A) an extraction process including dissolution of the RBC fraction, (B) a washing process, (C) a centrifugation process, or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the nucleic acid comprises either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

8. The method according to claim 7, wherein the nucleic acid is selected from the group consisting of normal RNA, normal DNA, tumor RNA, or tumor DNA.

9. The method according to any one of claims 1 to 8, wherein the very small embryoid stem cells from the whole blood sample are enriched by a method selected from the group consisting of flow cytometry, magnetic bead-based separation, filtration, microfluidic-based cell sorting, aptamer-based cell isolation, and buoyancy-activated cell sorting.

10. The method according to any one of claims 1 to 9, wherein the whole blood sample comprises at least one cell type selected from the group consisting of cancer stem cells and circulating tumor cells.

11. The method according to any one of claims 1 to 10, wherein the nucleic acid represents a genome.

12. The method according to any one of claims 1 to 10, wherein the nucleic acid represents a transcriptome.

13. The method according to any one of claims 1 to 10, wherein the nucleic acid represents an exome.

14. The method according to any one of claims 1 to 10, wherein the nucleic acid represents cDNA.

15. The method according to any one of claims 1 to 14, wherein the method provides information equivalent to all organ biopsies.

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