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1. (WO2019033203) METHOD FOR DETERMINATION OF CELLULAR MRNA
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CLAIMS

1. A method for mRNA analysis in cells, the method comprising :

introducing a first capture probe and a second capture probe into the cells, the first capture probe and the second capture probe each configured to be complementary to a respective section of target mRNA within the cells, wherein binding of the first and second capture probes to the respective sections of the target mRNA results in tagging of the cells and causes the first and second capture probes to form clusters with each other;

wherein the first capture probe and the second capture probe are each bound to magnetic nanoparticles (MNPs) that, when trapped within the tagged cells, cause the tagged cells to be susceptible to magnetic forces; and

introducing the cells into a device configured to magnetically capture tagged cells.

2. The method of claim 1 wherein the target mRNA is at least one of survivin, TMPRSS2/ERG, AR, AR-V7, PD1, PDL1, and PARP mRNA.

3. The method of claim 1 wherein the first capture probe has a first sequence and the second capture probe has a second sequence, and wherein : the first sequence of the first capture probe is 5' CAG TTC TTG AAT GTA GAG AT 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' GCA GGC GCA GCC CTC CAA GA 3';

the first sequence of the first capture probe is 5' GAT AAG GCT TCC TGC CGC GC 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' CAA CGA CTG GTC CTC ACT CA 3';

the first sequence of the first capture probe is 5' TGC TTT CAT GCA CAG GAA TT 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' CTG GAA TAA TGC TGA AGA GT 3'; or

the first sequence of the first capture probe is 5' CTG ATG AAG AGA AGC ATG TG 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' TGG GAG AAG AAT GAG AGG CT 3'.

4. The method of claim 1 wherein the cells are cancer cells.

5. The method of claim 4 wherein the cancer cells are prostate cancer cells.

6. The method of claim 1 wherein the device is a microfluidic device.

7. A system for analyzing mRNA in cells comprising :

a first capture probe and a second capture probe, the first capture probe and the second capture probe each configured to be complementary to a respective section of target mRNA within the cells, wherein binding of the first and second capture probes to the respective sections of the target mRNA results in tagging of the cells and causes the first and second capture probes to form clusters with each other;

wherein the first capture probe and the second capture probe are each bound to magnetic nanoparticles (MNPs) that, when trapped within the tagged cells, cause the tagged cells to be susceptible to magnetic forces; and

a device configured to magnetically capture tagged cells.

8. The system of claim 7 wherein the device is a microfluidic device.

9. The system of claim 8 wherein the microfluidic device further comprises a plurality of sorting portions defined in the microfluidic device, each sorting portion including a respective plurality of flow rate-reducing structures, wherein each sorting portion promotes capture of respective different cells exhibiting respective different amounts of susceptibility to magnetic attraction force.

10. The system of claim 9 wherein the microfluidic device comprises at least six sorting portions.

11. The system of claim 7 wherein the target mRNA is at least one of survivin, TMPRSS2/ERG, AR, AR-V7, PD1, PDL1, and PARP mRNA.

12. The system of claim 7 wherein the first capture probe has a first sequence and the second capture probe has a second sequence, and wherein :

the first sequence of the first capture probe is 5' CAG TTC TTG AAT GTA GAG AT 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' GCA GGC GCA GCC CTC CAA GA 3';

the first sequence of the first capture probe is 5' GAT AAG GCT TCC TGC CGC GC 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' CAA CGA CTG GTC CTC ACT CA 3';

the first sequence of the first capture probe is 5' TGC TTT CAT GCA CAG GAA TT 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' CTG GAA TAA TGC TGA AGA GT 3'; or

the first sequence of the first capture probe is 5' CTG ATG AAG AGA AGC

ATG TG 3'-Biotin-(TEG) and the second sequence of the second capture probe is Biotin-(TEG) -5' TGG GAG AAG AAT GAG AGG CT 3'.

13. The system of claim 7 wherein the cells are cancer cells.

14. The system of claim 13 wherein the cancer cells are prostate cancer cells.

15. A method for quantifying expression of target mRNA in cells of a sample, the method comprising :

determining an mRNA capture fraction of cells expressing the target mRNA in the sample; and

calculating an expression index (EI) for expression of the target mRNA in the sample by dividing the mRNA capture fraction by an average zone

parameter, the average zone parameter representing a zone in which cells having an average expression of the target mRNA is captured by a multi-zoned capture device;

wherein determining the mRNA capture fraction includes:

magnetically capturing cells that have been tagged with a targeted capture probe bound to a magnetic nanoparticle (MNP), the capture probe being configured to be complementary to a section of the target mRNA.

16. The method of claim 15, wherein determining the mRNA capture fraction further comprises :

determining a number of cells (N CP) captured in the sample using the targeted capture probe;

determining a number of cells (N NSP) captured in the sample by a nonspecific probe;

determining a total number of cells (NAb) in the sample; and

wherein the mRNA capture fraction is defined by an equation :

m RNA capture fraction = (N CP- N NSP) / NAb.

17. The method of claim 16, wherein determining NAb comprises determining a number of cells captured in the sample by anti-EpCAM .

18. The method of claim 16 further wherein calculating EI comprises calculating an equation :

EI = (m RNA capture fraction) / ZoneAve * 10

wherein ZoneAve is an index of an average capture zone in which cells having an average expression of the target mRNA are captured by the capture device.