MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

Summary

The protocol describes a tandem immunofluorescence hybridization assay with formalin-fixed paraffin-embedded tissue to detect and quantify microRNA and multiplexed proteins simultaneously.

Abstract

When using traditional probe-based in situ hybridization (ISH) imaging detection methods, microRNAs (miRNAs) are difficult to capture due to their size, which ranges from 19 to 25 nucleotides. However, as miRNAs are key epigenetic regulators that contribute to normal physiology and numerous pathological conditions, understanding the spatiotemporal behavior of miRNAs is important to unravel their function at the cellular and subcellular levels. This creates a clear need for adequate detection and visualization techniques. Therefore, we created MicroRNA Amplification and Recognition through Locked-nucleic-acid in situ hybridizatioN (MARLIN), which was developed and optimized utilizing multiplex immunofluorescence staining, and locked nucleic acid (LNA) probes targeted to a miRNA. This method can quantify miRNA with multiplexed protein expression, which allows the quantification of intracellular and extracellular miRNA within the tumor microenvironment (TME) with high sensitivity and reproducibility. The combined use of miRNA detection and protein capture assays can also provide a comprehensive understanding of cellular functions and regulatory mechanisms within that TME.

Introduction

microRNAs
Although microRNAs (miRNA) are incredibly small, ranging 19-25 nucleotides in length, these single-stranded RNA molecules are potent regulators in gene expression, modulating messenger RNAs (mRNAs) post-transcriptionally through sequence-specific interactions¹. In essence, miRNAs act as negative regulators by binding to target mRNAs, either preventing their translation or promoting their degradation through mRNA cleavage, depending on the degree of complementarity, which ultimately leads to decreased protein output from the targeted gene. While miRNAs are basically non-coding RNA, approximately 40% of all miRNAs....

Protocol

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissues of human ovarian tumor tissue sections from patients with HGSC. These samples were collected from previously untreated patients undergoing primary cytoreductive surgery for ovarian cancer. All samples and 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.

Results

When testing this protocol, it is important to have two serial sections of the same representative sample for accuracy and comparison. In this case, a High-Grade Ovarian Cancer (HGSC) sample was used to compare the results. A control slide (Figure 2A-B) that would only go through the miRNA staining and a test slide that would undergo both protein and miRNA staining (Figure 2C-J) were used.

Discussion

Though the importance of miRNAs is recognized and understood, observations of both normal development and disease states are currently hindered by the difficulty of studying miRNAs using standard methods. The scarcity of in situ methods led to the development of MARLIN (miRNA FISH assay). Given the homology and short length of miRNAs, it becomes a challenge to visualize their location and effect on post-transcriptomic regulation. This protocol development utilizes Locked Nucleic Acid Probes (LNA). LNAs are modif.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x TRIS-buffered Saline (TBS) pH 7.6	Thermo Fisher Scientific	J62662 K7	For intermittent washes between steps
1x Opal Antibody Diluent/Block	Akoya Biosciences	ARD1001EA	Protein blocking solution
1x Plus Automation Amplification Diluent	Akoya Biosciences	FP1609	Used to dilute Opal fluorophores
20x Concentrate Saline Sodium Citrate (SSC) Buffer	Sigma-Aldrich	S6639-1L	For miRNA washes after probe hybridization
4',6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific/ Invitrogen	EN62248 / 62248	For nuclear staining
ACD HybEZ II Hybridization System	ACD Biotechne	321711	Oven used to regulate temperature during miRNAscope protocol
Anti-CD8 (D8A8Y), Rabbit mAb	Cell Signaling Technologies	85336S	Example of primary antibody used to optimize this protocol
Anti-Digoxigenin (Mouse) HRP Conjugate	Revvity Health Sciences Inc	NEF832001EA	Secondary reagent used for miRNA detection
Anti-EpCAM (E6V8Y), Rabbit mAb	Cell Signaling Technologies	93790	Example of primary antibody used to optimize this protocol
Blocking Reagent Powder	Akoya Biosciences	FP1012	miRNA blocking solution
Ethyl Alcohol 100% (200 Proof)	Pharmco	111000200	For deparaffinization
EZ-AR1 Buffer	BioGeneX	HK521-XAK	Used for antigen retrieval of FFPE tissue sections at the protein step
EZ-AR1 Elegance Buffer	BioGeneX	HK546-XAK	Used for antigen retrieval of FFPE tissue sections at the miRNA probe step
EZ-Retriever IR System	BioGeneX	MW015-IR	Microwave used for antigen retrieval of FFPE tissue sections
Hydrogen Peroxide Solution 30%	Sigma-Aldrich	HX0640-5	For Tissue preparation. Helps to block endogenous peroxidase activity and to yield highly colored products
miR-181c-3p miRNA Probe	Qiagen	339112	Example of miRNA probe used to optimize this protocol
miRCURY LNA miRNA ISH Buffer Set (FFPE)	Qiagen	339450	Used to dilute miRNA probe
Opal 620	Akoya Biosciences	FP1495001KT	Fluorophore to identify primary antibody
Opal 690	Akoya Biosciences	FP1497001KT	Fluorophore to identify primary antibody
Opal Anti-Ms + Rb HRP	Akoya Biosciences	ARH1001EA	Secondary antibody used as a detection agent
Pierce 16% Formaldehyde (w/v), Methanol-free	Thermo Fisher Scientific	28908	To make 3.7% PFA for tissue preparation
ProLong Diamond Antifade	Thermo Fisher Scientific	P36970	Mounting media
RNAscope Protease Plus	ACD Biotechne	322331	To permeabilize tissue sections by partially digesting proteins & expose target miRNA molecules
RNAse-free water	Corning	46-000-CM	To clean and prep all equipment, and to use as a dilutant when necessary
Tween 20	Sigma-Aldrich	P1379-25ML	To make TBS-T
Tyramide Signal Amplification Plus Cyanine 3	Akoya Biosciences	NEL744001KT	Fluorophore to identify miRNA
U6 and Scramble miRNA Probes	Qiagen	339459	Optional control probes
Xylenes Histological Grade	Thermo Fisher Scientific	UN1307	For deparaffinization