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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 nucleotides3,4. Subsequently, the RNA-induced silencing complex (RISC) incorporates these miRNAs as single-stranded RNAs, which allows for their binding to the 3' untranslated region (3'-UTR) of their target mRNAs to suppress their expression3,5.

Within the last three decades, since they were first identified, miRNAs have emerged to master regulators of gene expression, whose own expression levels are tightly controlled6. Roles for miRNAs have been described in organ development7,8,9,10,11,12, maintenance of homeostasis13,14, as well as disease contexts that include neurological15,16,17,18,19, cardiovascular20, autoimmune conditions21,22, cancers23,24, and others25. The increasing appreciation for the relevance of miRNA expression patterns has brought forward the need for reliable detection methods of miRNA transcripts. Such methods include Real Time PCR, microarrays, Northern Blotting, in situ hybridization and others, which vary in the sensitivity, specificity, and quantitative power, predominantly due to the fact that miRNA transcripts are comprised of short and highly homologous sequences6.

We recently reported an important role for miRNA-182 in the development of the myocardial hypertrophy26, a condition describing the structural adaptation of the heart in response to elevated hemodynamic demands27,28. Cardiac hypertrophy is characterized by the increase in the myocardial mass, which, if associated with maladaptive remodeling29, can lead to increased risk for heart failure, a condition accounting for 8.5% of all deaths attributable to cardiovascular disease30.

Here, we describe our protocol that combines in situ hybridization with a digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe and immunostaining for the concurrent detection of miRNA and protein molecules on mouse heart tissue sections, in our model of cardiac hypertrophy.

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 Phosphate Buffered Saline (PBS) solution by dissolving 5 PBS tablets in 1 L of DEPC-treated ddH2O. Autoclave (121 °C).
  3. Prepare 0.1% non-ionic detergent/PBS (PBST) by diluting 1 mL of non-ionic detergent per 1 L of 1x PBS.
  4. Prepare 90%, 70%, 50% and 30% ethanol in DEPC-treated ddH2O.
  5. Prepare a 10 mg/mL stock solution of Proteinase K (ProK) in DEPC-treated ddH2O. Aliquot and store at -20 °C. Prepare a 20 μg/mL working solution of ProK by diluting 100 μL of ProK stock solution in 50 mL of DEPC-treated ddH2O. Make fresh before use.
  6. Prepare 4% paraformaldehyde (PFA) by adding 2 g of PFA in 50 mL of 1x PBS. Heat at 65 °C with occasional shaking, until dissolved. Aliquot and store at -20 °C.
    Caution: PFA is a known carcinogen, only handle in the fume hood.
  7. Prepare a 3 M NaCl solution by adding 87.8 g to 500 mL of ddH2O. Autoclave (121 °C).
  8. Prepare a 1 M MgCl2 solution by adding 20.3 g to 100 mL of ddH2O. Autoclave (121 °C).
  9. Prepare 1 M Tris pH 8.0 by dissolving 121.1 g in 800 mL of ddH2O. Adjust the pH to 8.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121 °C). Prepare a working solution of 50 mM Tris pH 8.0 by diluting 2.5 mL of 1 M Tris pH 8.0 in 47.5 mL of ddH2O.
  10. Prepare 1 M Tris pH 9.5 by dissolving 60.6 g in 400 mL of ddH2O. Adjust the pH to 9.5 with a few drops of 1 N HCl, bring the volume to 500 mL and autoclave (121 °C).
  11. Prepare 0.13 M 1-Methylimidazole pH 8.0 by diluting 1.6 mL of 1-Methylimidazole to 130 mL of DEPC-treated ddH2O. Adjust the pH to 8.0 with approximately 450 µL of 12 N HCl. Add 16 mL of 3 M NaCl and bring the volume to 160 mL. Make fresh before use.
    Caution: 1-Methylimidazole is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.
  12. Prepare 0.16 M N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) by diluting 176 µL of EDC to 10 mL of 0.13 M 1-Methylimidazole. Adjust the pH to 8.0 with approximately 100 µL of 12 N HCl. Make fresh before use.
    CAUTION: EDC is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.
  13. Prepare 1% H2O2 by diluting 1.5 mL of 30% H2O2 in 50 mL of 1x PBS. Make fresh before use.
  14. Prepare 20x SSC pH 7.0 by dissolving 175.3 g of NaCl and 88.2 g of sodium citrate (Na3C6H5O7) in 800 mL of ddH2O. Adjust the pH to 7.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121 °C).
    1. Prepare a working solution of 2x SSC pH 7.0 by diluting 20 mL of 20x SSC pH 7.0 in 180 mL of ddH2O.
    2. Prepare a working solution of 1x SSC pH 7.0 by diluting 2.5 mL of 20x SSC pH 7.0 in 47.5 mL of ddH2O.
    3. Prepare a working solution of 0.2x SSC pH 7.0 by diluting 5 mL of 2x SSC pH 7.0 in 45 mL of ddH2O.
  15. Prepare 1x hybridization solution by diluting 0.5 mL of 2x microRNA ISH buffer in 0.5 mL of DEPC-treated ddH2O. Make fresh before use.
  16. Prepare 200 mM stock solution of Levamisole by adding 250 mg to 5 mL of ddH2O. Aliquot and store at -20 °C.
  17. Prepare blocking solution for step 5 (10% sheep serum/1% BSA/0.2 mM Levamisol) by diluting 1 mL of sheep serum in 9 mL PBST, and adding 0.1 g of BSA. Add 10 μL of Levamisole before using. Make fresh before use.
  18. Prepare blocking solution for step 6 (10% goat serum/1% BSA) by diluting 1 mL of goat serum in 9 mL of PBST, and adding 0.1 g of BSA. Store at 4 °C and use within a week.
  19. Prepare prestaining solution by diluting 3.3 mL of 3 M NaCl, 5 mL of 1 M MgCl2,10 mL of 1 M Tris pH 9.5, 100 µL of Tween-20, 100 µL of Levamisole in 81.5 mL of ddH2O. Make fresh before use.
  20. Use nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indoly phosphate (BCIP) tablets to prepare 20 mL of Alkaline Phosphatase (AP) substrate solution according to the manufacturer's instructions. Add 20 μL of Levamisole. Make fresh before use and protect from light.
  21. Prepare KTBT solution by adding 4 g of Tris, 4.4 g of NaCl and 375 mg of KCl in 500 mL of ddH2O. Autoclave (121 °C).
  22. Optional: Prepare a DAPI working solution (1 μg/mL) by diluting 50 μL of DAPI stock solution (1 mg/mL) in 50 mL of 1x PBS.
    NOTE: Standard solutions such as 3 M NaCl, 1 M MgCl2, 1 M Tris-HCl, nuclease-Free ddH2O, 1x PBS and 20x SSC can be alternatively purchased. For this protocol, 50 mL glass Coplin jars or 20 mL polypropylene slide mailer jars have been used.

2. Tissue Preparation

NOTE: The mouse heart sections used here were prepared by Yale Pathology Tissue Services, from Formalin-Fixed/Paraffin-embedded tissue, cut at 5 μm and positioned on charged slides.

  1. Tissue rehydration.
    1. Warm the slides at 60 °C for 10 min, in the hybridization oven until paraffin is visibly melting.
    2. Incubate the slides in a glass Coplin jar containing 50 mL of commercially available clearing agent (see Table of Materials) for 10 min, twice.
    3. Incubate the slides in a glass Coplin jar containing 50 mL of 100% ethanol for 2 min, twice, followed by 90% ethanol for 2 min, 70% ethanol for 2 min, twice, 50% ethanol for 2 min and 30% ethanol for 2 min.
    4. Incubate the slides in a glass Coplin jar containing 50 mL of DEPC-treated ddH2O for 5 min.
    5. Incubate the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min.
  2. Tissue de-proteinating.
    1. Incubate slides in a glass Coplin jar containing 50 mL of 20 μg/mL ProK working solution, at 37 °C for 15 min in the hybridization oven.
      CAUTION: Under-digestion may result in poor or no signal development, while over-digestion may result in tissue degradation and development of background staining. Optimization may be required for this step (1–20 μg/mL ProK, 5–20 min incubation, RT-37 °C).
    2. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.
  3. Tissue fixation.
    1. Use a hydrophobic pen to draw a water repellent circle around the tissue on each slide.
    2. Apply 500 µL of 4% PFA solution to the length of each slide and incubate slides horizontally for 10 min, at RT.
    3. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.
    4. Wash the slides in a glass Coplin jar containing 50 mL of 0.13 M 1-methylimidazole for 10 min, at RT, twice.
    5. Apply 500 µL of 0.16 M EDC solution to the length of each slide and incubate slides horizontally for 1 h at RT, in a humidified chamber.
    6. Rinse the slides in a glass Coplin jar containing 50 mL of 50 mM Tris pH 8.0 at RT.
    7. Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice.
      NOTE: Both PFA and EDC fixation steps are required for miRNA crosslinking and should not be omitted.31
  4. Endogenous peroxidase block.
    1. Incubate slides in a glass Coplin jar containing 50 mL of 1% H2O2 for 30 min at RT.
    2. Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice.

3. Hybridization

  1. Preheat hybridization solution (500 µL per slide) at 50 °C in the hybridization oven, until the target temperature, is reached (10–15 min).
  2. Prepare the hybridization chamber by placing 2 pieces of filter or tissue paper in the bottom of the chamber and wetting the paper with 50% formamide/1x SSC.
    Caution: Formamide is harmful to mucous membranes, eyes and skin, only handle in the fume hood.
  3. Apply 200 µL of hybridization solution to the length of each slide. Incubate slides horizontally for 1–3 h at 50 °C, in a humidified chamber.
  4. Prepare the probe solution by mixing 0.5 µL stock probe (25 µM) in 250 µL ofhybridization solution (final concentration 50 nM) in a 1.5 mL tube.
    NOTE: Different miRNAs are expressed at different levels, so optimization regarding the probe concentration may be required for this step (1–50 nM), to avoid no signal or high background development.
  5. Apply 250 µL of probe solution to the length of each slide and place an RNase free coverslip on top. Avoid the formation of bubbles.
  6. Carefully seal the hybridization chamber with parafilm and incubate O/N at 50 °C.
    NOTE: The hybridization temperature should be set approximately 30 °C below the RNA melting temperature (Tm) of each probe. Optimization may be required. Here, 50 °C is used as the hybridization/wash temperature for miRNA-182, adjust accordingly for different probes.

4. Stringency Washes

  1. Prewarm 200 mL of 2x SSC at 50 °C in the hybridization oven, until the target temperature is reached (20–40 min).
  2. Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at 50 °C for 5 min, or until the coverslips loosen.
  3. Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at RT for 5 min, twice.
  4. Wash the slides in a glass Coplin jar containing 50 mL of 0.2x SSC at RT for 5 min.
  5. Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min.
  6. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min.
    NOTE: Use of RNase-free conditions is no longer necessary since the RNA-RNA hybrids are considered stable.

5. DIG Antibody Detection

  1. Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.
  2. Apply 500 µL of blocking solution to the length of each slide. Incubate slides horizontally for 30 min at RT, in a humidified chamber.
  3. Prepare the DIG antibody solution (1:1,000) by diluting 0.5 μL of DIG antibody in 500 µL ofblocking solution in a 1.5 mL tube.
    CAUTION: Optimization may be required for this step (1:500–1:2,000).
  4. Apply 500 µL of DIG antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.
  5. Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min, thrice.
  6. Wash the slides in a glass Coplin jar containing 50 mL of prestaining solution at RT for 5 min, twice.
  7. Incubate slides in a polypropylene slide mailer jar containing 20 mL ofAP substrate solution at 37 °C, for 6–24 h, protected from light.
    Caution: Color development should be checked every 2 h. Optimization may be required for this step.
  8. Wash the slides in a glass Coplin jar containing 50 mL of KTBT solution at RT for 5 min, twice.
  9. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min.

6. cTnT Antibody Detection

  1. Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.
  2. Apply 500 µL of blocking solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.
  3. Prepare the cTnT antibody solution (1:100) by diluting 2 μL of cTnT antibody in 200 μL ofblocking solution in a 1.5 mL tube.
  4. Apply 200 µL of cTnT antibody solution to the length of each slide. Incubate slides horizontally O/N at 4 °C, in a humidified chamber.
  5. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.
  6. Prepare the anti-rabbit-568 antibody solution (1:500) by diluting 0.5 μL of anti-rabbit-568 antibody in 250 μL ofblocking solution in a 1.5 mL tube.
  7. Apply 250 µL of anti-rabbit-568 antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.
  8. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.
  9. Optional: Apply 250 µL of DAPI working solution to the length of each slide, and incubate slides horizontally for 1 min at RT, protected from light.
  10. Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, twice.

7. Mounting and Imaging

  1. Mount the slides with 2–3 drops of mounting medium and a glass cover slip. Allow the slides to dry flat, O/N, at 4 °C, protected from light.
  2. Proceed to epifluorescent or confocal microscopy.

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 ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We would like to thank Athanasios Papangelis, for critical comments on the manuscript. FM is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M009424/1). IP is supported by the American Heart Association Scientist Development Grant (17SDG33060002).

Materials

NameCompanyCatalog NumberComments
DiethylpyrocarbonateSigma AldrichD5758DEPC
Phosphate buffered salineSigma AldrichP4417PBS
Tween-20American BioanalyticalAB02038non-ionic surfactant
HistoclearNational DiagnosticsHS-200
Proteinase K, recombinant, PCR GradeSigma Aldrich3115879001ProK
ParaformaldehydeSigma AldrichP6148PFA
Sodium ChlorideThermoFisherS271NaCl
Magnesium Chloride HexahydrateThermoFisherM33MgCl2
TrisSigma AldrichT6066
Hydrochloric Acid Solution, 1 NThermoFisherSA48HCl
Hydrochloric Acid Solution, 12 NThermoFisherS25358HCl
1-MethylimidazoleSigma Aldrich336092
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideSigma Aldrich39391EDC
Hydrogen peroxide solution H2O2Sigma Aldrich216763H2O2
Trisodium citrate dihydrateSigma AldrichS1804Sodium Citrate
miRCURY LNA miRNA ISH Buffer Set (FFPE)Qiagen339450scramble miRNA/U6 snRNA
miRCURY LNA mmu-miR-182 detection probeQIagenYD006157015'-DIG and 3'-DIG labelled
Levamisol hydrochlorideSigma Aldrich31742
Bovine Serum AlbuminSigma AldrichA9647BSA
NBT/BCIP TabletsSigma Aldrich11697471001NBT-BCIP
Potassium ChlorideThermoFisherP217KCl
DAPI solution (1mg/ml)ThermoFisher62248DAPI
Glass coverslipThermoFisher12-545EGlass coverslip
Plastic coverslipGrace Bio-LabsHS40 22mmX40mmX0.25mmRNA-ase free plastic coverslip
Anti-Digoxigenin-AP, Fab fragmentsSigma Aldrich11093274910DIG antibody
Hydrophobic barrier penVector LaboratoriesH-4000Pap pen
Anti-Cardiac Troponin T antibodyAbcamab92546cTnT antibody
Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568ThermoFisherA-11011anti-rabbit-568 antibody
Dako Fluorescence Mounting MediumDAKOS3023mounting medium
Sheep serumSigma AldrichS3772
Goat serumSigma AldrichG9023
Deionized FormamideAmerican BioanalyticalAB00600
Hybridization OvenThermoFisherUVP HB-1000 Hybridizer

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