Fully automated nucleic acid extraction methods for tissue samples

Patent metadata

Field	Value
Publication number	US-11390903-B2
Application number	US-201916442395-A
Country	US
Kind code	B2
Filing date	Jun 14, 2019
Priority date	Dec 22, 2016
Publication date	Jul 19, 2022
Grant date	Jul 19, 2022

Abstract

Automated methods for extracting nucleic acid from one or more tissue samples disposed on slides are disclosed. The methods utilize an automated slide staining apparatus that dispenses a plurality of nucleic acid extraction reagents onto the tissue sample, thus creating an extracted nucleic acid sample. The extracted nucleic acid sample may be used directly in downstream applications such as nucleic acid amplification or sequencing procedures, or may be further purified.

First claim

What is claimed is:

1. An automated method for extracting nucleic acid from tissue samples disposed on one or more slides in an automated staining machine, wherein the method comprises the following steps:
(a) treating the samples on the one or more slides with a deparaffinization reagent;
(b) treating the samples on the one or more slides with an antigen retrieval buffer;
(c) treating the samples on the one or more slides with a protease buffer; and
(d) heating the samples on the one or more slides to yield an eluate,
wherein the eluate comprises isolated nucleic acid from the respective sample, and
wherein the samples on the one or more slides are stained samples.

2. The method of claim 1 , further comprising removing the eluate from the samples on the one or more slides.

3. The method of claim 2 , further comprising purifying the isolated nucleic acid.

4. The method of claim 2 , further comprising performing nucleic acid amplification on the isolated nucleic acid.

5. The method of claim 3 , further comprising analyzing the isolated nucleic acid.

6. The method of claim 5 , wherein the analyzing comprises sequencing.

7. The method of claim 1 , wherein the nucleic acid is DNA.

8. The method of claim 1 , further comprising treating the samples on the one or more slides with guanidine thiocyanate after step (c).

9. The method of claim 8 , wherein the nucleic acid is RNA.

10. The method of claim 1 , wherein the samples on the one or more slides are FFPE samples, fresh samples, or frozen samples.

11. The method of claim 1 , wherein the samples are microdissected samples.

12. The method of claim 1 , wherein the stained samples are samples stained with haematoxylin and eosin (H&E), an immunohistochemistry dye, or an in situ hybridization dye.

13. The method of claim 1 , wherein the automated staining machine comprises one or more dispensers for dispensing the deparaffinization reagent, antigen retrieval buffer, and protease onto the samples on the one or more slides.

14. The method of claim 13 , wherein the automated staining machine further comprises a heating element for heating the samples on the one or more slides.

15. An automated method for extracting RNA from tissue samples disposed on one or more slides in an automated staining machine, wherein the method comprises the following steps:
(a) treating the samples on the one or more slides with a deparaffinization reagent;
(b) treating the samples on the one or more slides with an antigen retrieval buffer;
(c) treating the samples on the one or more slides with a protease;
(d) treating the samples on the one or more slides with guanidine thiocyanate; and
(e) heating the samples on the one or more slides to yield an eluate,
wherein the eluate comprises isolated nucleic acid from the respective sample, and
wherein the one or more slides are stained samples.

16. The method of claim 15 , further comprising removing the eluate from the samples on the one or more slides.

17. The method of claim 16 , further comprising purifying the isolated nucleic acid.

18. The method of claim 17 , further comprising performing nucleic acid amplification on the isolated nucleic acid.

19. The method of claim 18 , further comprising analyzing the isolated nucleic acid.

20. The method of claim 19 , wherein the analyzing comprises sequencing.

21. The method of claim 15 , wherein the samples on the one or more slides are FFPE samples, fresh samples, or frozen samples.

22. The method of claim 15 , wherein the samples are microdissected samples.

23. The method of claim 15 , wherein the stained samples are samples stained with H&E or an immunohistochemistry dye or an in situ hybridization dye.

24. An automated method for extracting nucleic acid from tissue samples disposed on one or more slides, wherein the method comprises the following steps:
(a) receiving the one or more slides comprising one or more tissue samples in an automated slide staining apparatus, wherein the one or more tissue samples are stained, wherein the automated slide staining apparatus is configured to automatically dispense a plurality of nucleic acid extraction reagents to the one or more tissue samples to treat the one or more tissue samples, respectively, the nucleic acid extraction reagents comprising a deparaffinization buffer, an antigen retrieval buffer, and a protease buffer;
(b) dispensing said reagents in a predetermined sequence such that the reagents come into contact with the one or more tissue samples on the one or more slides; and
(c) treating the one or more tissue samples on the one or more slides with the reagents, thereby resulting in one or more extracted nucleic acid samples comprising extracted nucleic acid.

25. The method of claim 24 , further comprising heating the tissue samples to elute extracted nucleic acid from the tissue sample.

26. The method of claim 24 , further comprising removing the one or more extracted nucleic acid samples from the one or more slides.

27. The method of claim 26 , further comprising purifying the nucleic acid from one or more extracted nucleic acid samples.

28. The method of claim 27 , further comprising analyzing the nucleic acid from one or more extracted nucleic acid samples.

29. The method of claim 28 , wherein the analyzing comprises performing nucleic acid amplification.

30. The method of claim 28 , wherein the analyzing comprises nucleic acid sequencing.

31. The method of claim 24 , wherein the extracted nucleic acid is DNA.

32. The method of claim 24 , wherein the reagents further comprise guanidine thiocyanate.

33. The method of claim 32 , wherein the extracted nucleic acid is RNA.

34. The method of claim 24 , wherein the one or more tissue samples are fresh, frozen, formalin fixed, or paraffin embedded.

35. A method for isolating nucleic acid from a sample on a slide, wherein the method comprises the following steps:
(a) placing the sample on the slide on an automated staining machine, wherein the sample is a stained sample;
(b) running an extraction protocol on the automated staining machine, wherein the extraction protocol instructs the automated staining machine to dispense reagents, wherein the reagents comprise: (i) a deparaffinization buffer, (ii) an antigen retrieval buffer, (iii) and a protease buffer, wherein the reagents are dispensed on the slide in a predetermined sequence, such that said reagents come into contact with the sample on the slide, thereby resulting in an eluate on the sample on the slide, and wherein the eluate comprises an isolated nucleic acid sample comprising isolated nucleic acid.

36. The method of claim 35 , further comprising heating the sample to yield the eluate on the sample on the slide.

37. The method of claim 35 , further comprising removing the eluate on the sample on the slide.

38. The method of claim 37 , further comprising purifying the isolated nucleic acid.

39. The method of claim 38 , further comprising performing nucleic acid amplification on the isolated nucleic acid.

40. The method of claim 37 , further comprising analyzing the isolated nucleic acid.

41. The method of claim

Assignees

• Ventana Med Syst Inc

Inventors

• Day William
• Peccarelli Megan C

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title