Method for extracting nuclei or whole cells from formalin-fixed paraffin-embedded tissues

Patent metadata

Field	Value
Publication number	US-2022411783-A1
Application number	US-201917284542-A
Country	US
Kind code	A1
Filing date	Oct 11, 2019
Priority date	Oct 12, 2018
Publication date	Dec 29, 2022
Grant date	—

Abstract

The subject matter disclosed herein is generally directed to isolating single cells and nuclei from tissue samples for use in the analysis of single cells from archived biological samples. The subject matter disclosed herein is directed to isolating single cells and nuclei from formalin-fixed paraffin-embedded (FFPE) tissues. The subject matter disclosed herein is also directed to isolating single nuclei that preserve ribosomes or ribosomes and rough ER from frozen tissues. The subject matter disclosed herein is also directed to therapeutic targets, diagnostic targets and methods of screening for modulating agents.

First claim

What is claimed is:

1 . A method of recovering nuclei or whole cells from a formalin-fixed paraffin-embedded (FFPE) tissue comprising:
a. dissolving paraffin from a FFPE tissue sample in a solvent, preferably the solvent is selected from the group consisting of xylene and mineral oil, wherein the tissue is dissolved at a temperature between 4 C to 90 C, preferably room temperature (20 to 25 C) for recovering whole cells and 90 C for recovering nuclei; b. rehydrating the tissue using a gradient of ethanol from 100% to 0% ethanol (EtOH); c. transferring the rehydrated tissue to a volume of a first buffer comprising a buffering agent, a detergent and an ionic strength between 100 mM and 200 mM, optionally the first buffer comprises protease inhibitors or proteases and/or BSA; d. chopping or dounce homogenizing the tissue in the buffer; and e. removing debris by filtering and/or FACS sorting.

2 . The method of claim 1 , further comprising isolating nuclei or cell types by FACS sorting.

3 . The method of claim 1 , wherein dissolving paraffin from a FFPE tissue sample, comprises incubating at least one time in xylene, at room temperature (RT), for about 10 minutes each, and wherein xylene is removed at each change.

4 . The method of claim 3 , further comprising washing the tissue at least two times with xylene for about 10 min each, wherein the washes are performed at room temperature (RT), 90 C, or at least one time at room temperature (RT) and at least one time at 90 C, wherein xylene is removed at each change.

5 . The method of claim 1 , wherein dissolving paraffin from a FFPE tissue sample, comprises incubating at least twice in about 5 ml xylene per 30-100 mg FFPE tissue sample, at room temperature, for about 10 minutes each, wherein xylene is removed at each change.

6 . The method of claim 5 , further comprising washing the tissue with xylene at 37 C for about 10 min.

7 . The method of claim 6 , further comprising cutting the tissue into two or more pieces and washing at least one piece of the tissue with xylene at 37 C for about 10 min.

8 . The method of claim 1 , wherein dissolving paraffin from a FFPE tissue sample, comprises incubating at least three times in xylene, at room temperature, for about 10 minutes each, and wherein xylene is removed at each change.

9 . The method of claim 8 , further comprising washing the tissue three additional times with xylene for about 10 min each, wherein the first wash is at room temperature and the second and third washes are at 90 C, and wherein xylene is removed at each change.

10 . The method of claim 1 , wherein rehydrating the tissue comprises a step gradient of ethanol (EtOH) and the tissue is incubated between 1 to 10 minutes at each step.

11 . The method of claim 10 , wherein the step gradient comprises incubating the tissue for about 2 minutes each in successive washes of 95%, 75%, and 50% ethanol (EtOH).

12 . The method of any of the preceding claims, wherein after rehydrating the tissue the method further comprises placing the tissue samples on ice or on a device capable of maintaining the tissue between 4 and 10 C, wherein all subsequent steps are performed at a temperature between 4 and 10 C.

13 . The method of any of the preceding claims, wherein after the step of dissolving paraffin from the tissue or rehydrating the tissue the method further comprises dividing the tissue, preferably in half.

14 . The method of claim 1 , wherein the first buffer comprises a detergent selected from the group consisting of NP40, CHAPS and Tween-20.

15 . The method of claim 14 , wherein the NP40 concentration is about 0.2%.

16 . The method of claim 14 , wherein the Tween-20 concentration is about 0.03%.

17 . The method of claim 14 , wherein the CHAPS concentration is about 0.49%.

18 . The method of claim 1 , wherein the first buffer is selected from the group consisting of CST, TST, NST and NSTnPo.

19 . The method of claim 1 , wherein after the step of chopping or dounce homogenizing the method further comprises centrifuging, preferably, the sample is centrifuged at about 500 g for about 5 min, and resuspending the sample in a second buffer comprising a buffering agent and an ionic strength between 100 mM and 200 mM, optionally the second buffer comprises protease inhibitors.

20 . The method of claim 19 , wherein the second buffer is ST, optionally comprising protease inhibitors.

21 . The method of claim 1 , wherein the sample is filtered through a 40 uM filter.

22 . The method of claim 21 , further comprising washing the filtered sample in the first buffer.

23 . The method of claim 22 , further comprising filtering the sample through a 30 uM filter.

24 . The method of claim 1 , wherein after the step of chopping or dounce homogenizing the method further comprises adding an additional 2 volumes of the first buffer (3 volumes total) and filtering the sample through a 40 uM filter.

25 . The method of claim 24 , further comprising adding an additional three volumes of the first buffer (6 volumes total), centrifuging, preferably, the sample is centrifuged at about 500 g for about 5 min, and resuspending the sample in a second buffer comprising a buffering agent and an ionic strength between 100 mM and 200 mM, optionally the second buffer comprises protease inhibitors.

26 . The method of claim 25 , wherein the second buffer is ST, optionally comprising protease inhibitors.

27 . The method of any of the preceding claims, further comprising reversing cross-linking in the tissue sample before or during any step of the method.

28 . The method of claim 27 , wherein reversing cross-linking comprises proteinase digestion.

29 . The method of claim 28 , wherein the proteinase is proteinase K or a cold-active protease.

30 . The method of any of the preceding claims, further comprising adding a reagent that stabilizes RNA to the tissue sample before or during any step of the method.

31 . The method of any of the preceding claims, further comprising lysing recovered cells or nuclei and performing reverse transcription.

32 . The method of claim 31 , wherein the reverse transcription is performed in individual reaction vessels.

33 . The method of claim 31 , wherein the reaction vessels are wells, chambers, or droplets.

34 . The method of any of the preceding claims, further comprising performing single cell, single nucleus or bulk RNA-seq, DNA-seq, ATAC-seq, or ChIP on the recovered nuclei or whole cells.

35 . The method of any of the preceding claims, further comprising staining the recovered cells or nuclei.

36 . The method of claim 35 , wherein the stain comprises ruby stain.

37 . A method of recovering nuclei and attached ribosomes from a tissue sample comprising:
a. chopping the tissue sample at between 0-4° C. in a nuclear extraction buffer comprising Tris buffer, a detergent and salts; and b. filtering the sample through a filter between 30-50 uM, preferably 40 uM, and optionally washing the filter with fresh nuclear extraction buffer, wherein the nuclei are present in the supernatant passed through the filter.

38 . The method of claim 37 , wherein the nuclear extra

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Massachusetts Gen Hospital

Inventors

• Regev Aviv
• Mccabe Cristin
• Drokhlyansky Eugene
• Melnikov Alexandre
• Rozenblatt-Rosen Orit
• Smillie Christopher S
• Xavier Ramnik J
• Ashenberg Orr
• Eraslan Gökcen
• Chung Hattie

Classifications

• C12N5/0081

  Purging biological preparations of unwanted cells · CPC title

• G01N2001/2873

  Cutting or cleaving · CPC title

• G01N1/34

  Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)} · CPC title

• C12N15/1017 Primary

  by filtration, e.g. using filters, frits, membranes · CPC title

• G01N1/286

  involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F) · CPC title