RNA In situ Hybridization with Sequential Protein Immunofluorescence in Tandem Assay

Summary

This protocol details RNA in situ hybridization with sequential protein immunofluorescence in tissue on an automated platform to characterize targeted cellular-level spatial multiple omics.

Abstract

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest proteins, must operate in harmony and coordination within the human body. As such, the importance of approaches that integrate cellular-level spatial investigations across multiple omics is paramount to understanding cell-cell interactions and the progression of disease. Dissecting the proteome and transcriptome in the same spatial assay helps us understand how not only what a cell is being instructed to carry out (RNA), but also how it is executing those instructions in the context of the microenvironmental niche it finds itself in (protein). This manuscript is focused on integrating sequential immunofluorescence (SeqIF) with RNA in situ hybridization (ISH) with an on-tissue microfluidics driven system for high-throughput protein and RNA investigation in a spatial context (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run with additional protein targets possible to be added via sequential runs. This method provides a sequential targeted multiomics platform that does not require consideration of fluorophore compatibility and extensive optimization for higher plexing. This allows one to understand what messages the cell is priming or is sending into its microenvironment. This targeted approach helps to validate whole transcriptome methods while examining the interactions between the RNA and proteins in a more precise manner.

Introduction

The complex functioning of cells within the human body relies on an intricate interplay of signals, from small RNA molecules to large proteins, all working in coordination¹. This interaction underscores the importance of integrative approaches that examine cellular-level spatial interactions across various omics layers^2,3. Such methodologies are essential for understanding the complexities of cell-cell interactions and disease progression. Many traditional methods, such as flow cytometry⁴ and single-cell sequencing⁵, cell isolates are used....

Protocol

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissue sections collected from treatment-naive high-grade serous ovarian carcinoma patients that underwent primary cytoreductive surgery. All clinical data were obtained from the ovarian cancer repository of the Department of Gynecologic Oncology and Reproductive Medicine under protocols approved by the University of Texas MD Anderson's Institutional Review Board. Written informed consent from the patients was obtained by front desk personnel, and the studies were conducted in accordance with recognized ethical guidelines.

NOTE: All reagents, unless otherwise stated, are diluted....

Results

As an example of this method, we ran the following seqRNA-ISH (Table 1) + SeqIF (Table 2 and Table 3) on two slides from a human high-grade serous ovarian carcinoma.

Figure 2 illustrates the overlay of seqRNA-ISH+seqIF on a sample of ovarian tumor tissue. Structural protein markers such as collagen 1 are utilized to identify tumor regions, while phenotypic and functional markers like CD33, CD8, and Granzyme B (GZM.......

Discussion

The most critical step in preparing and handling samples is lowering RNAse contamination. This can be achieved by utilizing masks to prevent contamination from breath and by wiping work surfaces down with an RNase decontamination solution and 70% ethanol made with nuclease-free water. For FFPE slides, it is best to cut multiple, adjacent slides from FFPE blocks for seqRNA-ISH+seqIF and store them at 4 °C with desiccant. For quality control, in addition to testing the DV200 of tissue blocks, it is recommended to run .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4',6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific/ Invitrogen	EN62248 / 62248	For nuclear staining (product used during this study, 62247 has been discontinued)
50mL Conical Sterile Polypropylene Centrifuge Tubes	Thermo Fisher Scientific	339652	Used for aliquots & mixed secondary solutions
70% Sterile Isopropanol Alcohol	Texwipe	TX3270	For surface decontamination
Antibodies	Various	Various	Used for primary targets, varies based on channel used
APOE - Host: Mouse, Clone: 960318	R&D	MAB41443-100	(note: Specific to this study) Primary protein Antibody
aSMA - Host: Mouse, Clone: 1A4	CST	69319SF	(note: Specific to this study) Primary protein Antibody
Autocut microtome	Leica	14051956472	Sectioning of tissue
CD10 - Host: Rabbit, Clone: E5P7S	CST	65534S	(note: Specific to this study) Primary protein Antibody
CD11c - Host: Rabbit, Clone: D3V1E	CST	93233SF	(note: Specific to this study) Primary protein Antibody
CD163 - Host: Rabbit, Clone: D6U1J	CST	93498S	(note: Specific to this study) Primary protein Antibody
CD163 - Host: Rabbit, Clone: ja51-30	Invitrogen	ma5-32684	(note: Specific to this study) Primary protein Antibody
CD20 - Host: Mouse, Clone: L26	Bethyl	A500-017ACF	(note: Specific to this study) Primary protein Antibody
CD31 - Host: Mouse, Clone: 3F8E2	ProteinTech	66065-2-Ig	(note: Specific to this study) Primary protein Antibody
CD33 - Host: Rabbit, Clone: BLR061G	Bethyl	A700-061CF	(note: Specific to this study) Primary protein Antibody
CD4 - Host: Rabbit, Clone: EPR6855	Abcam	ab181724	(note: Specific to this study) Primary protein Antibody
CD45 - Host: Rabbit, Clone: BL-178-12C7	Bethyl	A700-012	(note: Specific to this study) Primary protein Antibody
CD45RO - Host: Mouse, Clone: UCHL1	Bethyl	A500-020ACF	(note: Specific to this study) Primary protein Antibody
CD56 - Host: Rabbit, Clone: BLR152J	Bethyl	99746	(note: Specific to this study) Primary protein Antibody
CD66b - Host: Rabbit, Clone: BLR111H	Invitrogen	MA5-44413	(note: Specific to this study) Primary protein Antibody
CD68 - Host: Mouse, Clone: KP-1	Biolegend	916104	(note: Specific to this study) Primary protein Antibody
CD8 - Host: Mouse, Clone: C8/144B	Biolegend	372902	(note: Specific to this study) Primary protein Antibody
CD86 - Host: Rabbit, Clone: E2G8P	CST	76755SF	(note: Specific to this study) Primary protein Antibody
Col 1A1 - Host: Mouse, Clone: E3E1X	CST	66948S	(note: Specific to this study) Primary protein Antibody
DNA LoBind Tubes 2mL	Eppendorf	22431048	Used for mixed RNA Probe solutions
Drierite 21005 Indicating Desiccant	Cole-Palmer	21005	Desiccant for Desiccator
Edge-Rite Microtome Blades	Thermo Fisher Scientific / Invitrogen	4280L	Histology - Used when sectioning samples
Ethyl Alcohol 100% (200 Proof)	Pharmco	111000200	For deparaffinization
EZ-AR2 Elegance Buffer	BioGeneX	HK547-XAK	Used for antigen retrieval of FFPE tissue sections
FOXP3 - Host: Rabbit, Clone: Polyclonal	Bethyl	A700-034CF	(note: Specific to this study) Primary protein Antibody
Goat anti-Mouse IgG (H+L) Alexa Fluor 555	Thermo Fisher Scientific/ Invitrogen	A-21425	(note: Specific to this study) F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555
Goat anti-Mouse IgG (H+L) Alexa Fluor 647	Thermo Fisher Scientific/ Invitrogen	A-48289/A-21237	(note: Specific to this study) F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647
Goat anti-Rabbit IgG (H+L) Alexa Fluor 555	Thermo Fisher Scientific/ Invitrogen	A-21430	(note: Specific to this study) F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555
Goat anti-Rabbit IgG (H+L) Alexa Fluor 647	Thermo Fisher Scientific/ Invitrogen	A-21246	(note: Specific to this study) F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647
GZMB - Host: Rabbit, Clone: Polyclonal	ProteinTech	13588-1-AP	(note: Specific to this study) Primary protein Antibody
Hs-POLR2A-T1	ACD Biotechne	310457-T1	Human low expressing probe
Hs-PPIB-T5	ACD Biotechne	313907-T5	Human mid expressing probe control
Hs-UBC-T9	ACD Biotechne	310047-T9	Human high expressing control
Hydrogen Peroxide Solution 30%	Sigma-Aldrich	HX0640-5	For Tissue preparation. Helps to block endogenous peroxidase activity and to yield highly colored products. To make 3% Hydrogen Peroxide Solution
Ker8/18 - Host: Mouse, Clone: Polyclonal	ProteinTech	66187-1-PBS	(note: Specific to this study) Primary protein Antibody
LRP5 - Host: Rabbit, Clone: HPA030505	Sigma	HPA030505-100UL	(note: Specific to this study) Primary protein Antibody
Lunaphore 20x Multistaining Buffer	Bio-Techne	BU06	Used as dilutent and for washes for the COMET
Lunaphore COMET Chip	Bio-Techne	MK03	mcirofluidics chip for COMET instrument
Lunaphore COMET	Bio-Techne	CM10-S	Seq-IF autostainer
Lunaphore Elution Buffer Kit	Bio-Techne	BU07-L	Solution 1 & Solution 2 for the elution steps of the COMET
Lunaphore Imaging Buffer Kit	Bio-Techne	BU09	Solute & Solvent used for imaging during the COMET run
Lunaphore Quenching Buffers	Bio-Techne	BU08-L	Solution 1 & Solution 2 for the quenching steps of the COMET
Micropipette	Various	N/A	for reagent preparation
Periostin - Host: Mouse, Clone: 1A11A3	ProteinTech	66491-1-PBS	(note: Specific to this study) Primary protein Antibody
Pierce 16% Formaldehyde (w/v), Methanol-free	Thermo Fisher Scientific	28908	To make 3.7% PFA for tissue preparation
Probe 320102 in (T2 channel)	ACD Biotechne	300040	Negative control DapB in T2
Probe 320102 in (T3 channel)	ACD Biotechne	300040	Negative control DapB in T3
Probe 320102 in (T4 channel)	ACD Biotechne	300040	Negative control DapB in T4
RNAscope HiPlex Cleaving Stock Solution	Bio-Techne	P/N 324399	Reagent for cleaving during RNAScope assay
RNAscope HiPlex Pro for COMET 12-plex, 20-slide Kit	Bio-Techne	322075	Reagents & Buffers for RNAScope
RNAscope HiPlex Probe- Hs-APOE-T2	ACD Biotechne	433093-T2	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-APOE-T2
RNAscope HiPlex Probe- Hs-ARG1-T4	ACD Biotechne	401581-T4	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-ARG1-T4
RNAscope HiPlex Probe- Hs-CD274-T1	ACD Biotechne	600863-T1	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CD274-T1
RNAscope HiPlex Probe- Hs-CD40-T6	ACD Biotechne	445973-T6	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CD40-T6
RNAscope HiPlex Probe- Hs-CXCL1-01-T12	ACD Biotechne	1256473-T12	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CXCL1-01-T12
RNAscope HiPlex Probe- Hs-GZMB-T7	ACD Biotechne	468453-T7	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-GZMB-T7
RNAscope HiPlex Probe- Hs-IFNG-T3	ACD Biotechne	310503-T3	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IFNG-T3
RNAscope HiPlex Probe- Hs-IL23a-T8	ACD Biotechne	562853-T8	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IL23a-T8
RNAscope HiPlex Probe- Hs-IL6-T9	ACD Biotechne	400883-T9	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IL6-T9
RNAscope HiPlex Probe- Hs-TNFa-T11	ACD Biotechne	310423-T11	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-TNFa-T11
RNAscope HiPlex Probe- Hs-VEGFa-T5	ACD Biotechne	423163-T5	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-VEGFa-T5
RNAscope HiPlex Probe- Hs-VISTA-T10	ACD Biotechne	491513-T10	(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-VISTA-T10
RNAscope HiPlex12 CS Negative Control Probe	ACD Biotechne	324347	Full 12 plex negative control panel recommended by the manufacturer. DapB in T1-T12.
RNAscope HiPlex12 CS Positive Control Probe-Hs	ACD Biotechne	324317	Full 12 plex Human positive control panel recommended by the manufacturer. Hs -RTU for following housekeeping gene in channels T1 to T12: Polr2a, PPIB, UBC, HPRT1, TUBB, RPL28, RPL5, B2M, ACTB, LDHA-O1, RPLP0-X-RPLP0P2, GAPDH.
RNAse-free water	Corning	46-000-CM	To clean and prep all equipment, and to use as a dilutant when necessary
RNaseZap RNase Decontamination Solution	Thermo Fisher Scientific	AM9782	A surface decontamination solution that destroys RNases
RNeasy FFPE Kit	Qiagen	73504 & 19093	For RNA Extraction & DV200 measurement
Slide Mailer	Simport Scientific	M9504MA	for storing slides in liquid buffer.
Surgical Design General Purpose Industrial Razor Blade	Thermo Fisher Scientific / Invitrogen	13-812-236	Histology - Used when sectioning samples
TOX 1/2 - Host: Rabbit, Clone: E613Q	CST	62886SF	(note: Specific to this study) Primary protein Antibody
Xylene Histological Grade	Thermo Fisher Scientific	UN1307	For deparaffinization