Focal gene expression profiling of stained FFPE tissues with spatial correlation to morphology

1. A method for detecting a nucleic acid sequence from a selected area of a sample in situ, comprising in any order:

imaging the sample for the presence or absence of an analyte;

selecting an area of the sample less than 2 mm 2 based on the imaging;

detecting a target nucleic acid sequence having a downstream region (DR) and an upstream region (UR), by

(1) contacting at least the selected area of the sample with a downstream detector oligo (DDO) comprising a DR′ portion that is complementary to the DR, and an upstream detector oligo (UDO) comprising a UR′ portion that is complementary to the UR,

(2) ligating the DR′ and UR′ if both are specifically hybridized to the DR and UR of a target sequence, and

(3) eluting the ligation products from the selected area by addition of a basic elution fluid,

whereby the ligation product indicates the presence of the target sequence in the selected area.

2. The method of claim 1 , wherein the area is selected is a morphological feature.

3. The method of claim 1 , wherein the sample is from an FFPE.

4. The method of claim 1 , further comprising the step of deparaffinizing the sample.

5. The method of claim 1 , further comprising the step of analyte retrieval.

6. The method of claim 5 , wherein the analyte is an antigen or nucleic acid.

7. The method of claim 1 , wherein the sample is imaged by histological stains.

8. The method of claim 1 , wherein the sample is imaged by immunostaining.

9. The method of claim 1 , wherein the sample is imaged with a histological stain and by immunostaining.

10. The method of claim 1 , wherein the sample is dried after performing a step.

11. The method of claim 1 , wherein a wash step is performed after detection steps (1) or (2).

12. The method of claim 1 , wherein the selected area is less than 0.2, 0.02, or 0.002 mm 2 .

13. The method of claim 1 , wherein the target sequence is a portion of a cancer marker.

14. The method of claim 1 , further comprising the step of reimaging the selected area, to verify collection of ligation products.

15. A method for detecting a neoplastic state of a cell by performing the method of claim 1 where a first cancer marker sequence is detected in the cell.

16. The method of claim 15 , wherein the number of ligation products detected for a second cancer marker sequence is less than 10%, 1%, 0.01%, or 0.05% of the number of ligation products detected for the first cancer marker sequence.

17. The method of claim 16 , wherein a cancer marker is detected a neoplastic cell and not detected in a normal cell, providing absolute specificity.

18. A method for generating a gene expression profile for a selected area, comprising performing the method of claim 1 for a plurality of target sequences.

19. A method for detecting a neoplastic state of a cells in a tissue by performing the method of claim 18 for a plurality cancer marker sequences on cells in two separate areas of the tissue.

20. A method for diagnosing a disease state by performing the method of claim 1 , wherein the target sequence is detected in the area of a morphological feature.

21. A kit comprising the detector oligos of claim 1 , a basic elution fluid, and a stain.

22. The method of claim 1 , wherein elution step (3) comprises applying the basic elution fluid to the selected area of the sample in situ.

23. The method of claim 1 , wherein elution step (3) comprises applying the basic elution fluid to the selected area of the sample using a capillary with fluidic control.

24. The method of claim 1 , wherein elution step (3) comprises recovering the ligation products from the selected area of the sample in situ.

25. The method of claim 1 , wherein elution step (3) comprises transferring the eluted ligation products using a capillary with fluidic control.

26. The method of claim 1 , wherein elution step (3) comprises transferring the eluted ligation products to a container.