Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Summary

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Abstract

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Introduction

micro-RNAs (miRNAs) are short (18–25 nucleotides), single-stranded, noncoding RNAs that function as negative regulators of gene expression at the post-transcriptional level by inhibiting messenger RNA (mRNA) translation and/or promoting mRNA degradation¹. miRNAs are transcribed from introns or exons of coding or noncoding genes and are cleaved in the nucleus by DROSHA, to precursor miRNAs (pre-miRNAs), which are short stem-loop structures of 70 nucleotides². Following cytoplasmic export, pre-miRNAs are further processed by DICER into mature miRNAs that span 18–25 nucleotides^3,

Protocol

Mouse heart tissue samples for this study were obtained in compliance with the relevant laws and institutional guidelines and were approved by Yale University Institutional Animal Care and Use Committee.

1. Solution Preparation

1. Prepare RNase free ddH₂O, by treating 5 L of ddH₂O with 5 mL of 0.1% diethylpyrocarbonate (DEPC) overnight (O/N), at room temperature (RT). Autoclave (121 °C) to deactivate the DEPC. Use DEPC-treated ddH₂O for the preparation of downstream solutions as indicated.
   CAUTION: DEPC is a known carcinogen, only handle in the fume hood.
2. Prepare 1x Phosph....

Results

miRNA in situ hybridization was optimized on mouse heart sections using a scramble miRNA and U6-snRNA, which served as negative and positive controls respectively. Positive staining is indicated in blue, while the negative staining is indicated by the lack of color development (Figure 1A-1B). Cardiomyocyte specific expression of miRNA-182 was assessed in heart sections from control and PlGF overexpressing mice. The mice carrying the .......

Discussion

miRNA transcript detection can be achieved through different techniques that vary in sensitivity, specificity and quantitative power. Here, we demonstrate the coupling of miRNA in situ hybridization with immunostaining and describe a protocol that allows for concurrent assessment of the expression levels of miRNA and protein molecules, on the same heart sections. We first show how to perform in situ hybridization of DIG-labeled LNA miRNA probes on paraffin embedded heart sections. Next, we describe how .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Diethylpyrocarbonate	Sigma Aldrich	D5758	DEPC
Phosphate buffered saline	Sigma Aldrich	P4417	PBS
Tween-20	American Bioanalytical	AB02038	non-ionic surfactant
Histoclear	National Diagnostics	HS-200
Proteinase K, recombinant, PCR Grade	Sigma Aldrich	3115879001	ProK
Paraformaldehyde	Sigma Aldrich	P6148	PFA
Sodium Chloride	ThermoFisher	S271	NaCl
Magnesium Chloride Hexahydrate	ThermoFisher	M33	MgCl2
Tris	Sigma Aldrich	T6066
Hydrochloric Acid Solution, 1 N	ThermoFisher	SA48	HCl
Hydrochloric Acid Solution, 12 N	ThermoFisher	S25358	HCl
1-Methylimidazole	Sigma Aldrich	336092
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride	Sigma Aldrich	39391	EDC
Hydrogen peroxide solution H2O2	Sigma Aldrich	216763	H2O2
Trisodium citrate dihydrate	Sigma Aldrich	S1804	Sodium Citrate
miRCURY LNA miRNA ISH Buffer Set (FFPE)	Qiagen	339450	scramble miRNA/U6 snRNA
miRCURY LNA mmu-miR-182 detection probe	QIagen	YD00615701	5'-DIG and 3'-DIG labelled
Levamisol hydrochloride	Sigma Aldrich	31742
Bovine Serum Albumin	Sigma Aldrich	A9647	BSA
NBT/BCIP Tablets	Sigma Aldrich	11697471001	NBT-BCIP
Potassium Chloride	ThermoFisher	P217	KCl
DAPI solution (1mg/ml)	ThermoFisher	62248	DAPI
Glass coverslip	ThermoFisher	12-545E	Glass coverslip
Plastic coverslip	Grace Bio-Labs	HS40 22mmX40mmX0.25mm	RNA-ase free plastic coverslip
Anti-Digoxigenin-AP, Fab fragments	Sigma Aldrich	11093274910	DIG antibody
Hydrophobic barrier pen	Vector Laboratories	H-4000	Pap pen
Anti-Cardiac Troponin T antibody	Abcam	ab92546	cTnT antibody
Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568	ThermoFisher	A-11011	anti-rabbit-568 antibody
Dako Fluorescence Mounting Medium	DAKO	S3023	mounting medium
Sheep serum	Sigma Aldrich	S3772
Goat serum	Sigma Aldrich	G9023
Deionized Formamide	American Bioanalytical	AB00600
Hybridization Oven	ThermoFisher	UVP HB-1000 Hybridizer