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.
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.
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.
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
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.
3. Hybridization
4. Stringency Washes
5. DIG Antibody Detection
6. cTnT Antibody Detection
7. Mounting and Imaging
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 PlGF transgene under a αMHC promoter develop cardiac hypertrophy, secondary to increased angiogenesis, within 6 weeks of transgene activation26. We performed in situ hybridization and found increased expression of miRNA-182 in the hearts of PlGF mice, compared to controls (Figure 2A-2H), indicated by the blue staining. To determine which cell types express miRNA-182, we then performed immunostaining for cTnT on the same sections. We found miRNA-182 co-localized with the cTnT-positive cardiomyocyte cells, as well as DAPI stained nuclei, in both control and PlGF mouse heart sections (Figure 2C-2H), indicated by the red and blue staining respectively. These images were with a 20X objective.
Figure 1: Negative and Positive in situ hybridization staining. (A) In situ hybridization for scramble miRNA (25 nM) in control mouse heart sections. (B) In situ hybridization for U6-snRNA (25 nM) in control mouse heart sections. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 2: Cardiomyocyte specific expression of miRNA-182 in control and PlGF mouse hearts. (A-B) In situ hybridization for miRNA-182 in control and PlGF mouse heart sections. (C-D) Immunostaining for cTnT in control and PlGF mouse heart sections that have been previously stained for miRNA-182. (E-F) DAPI counterstaining for nuclear localization in control and PlGF mouse heart sections that have been previously stained for miRNA-182. (G-H) Merged images of miRNA-182 (deep blue), cTnT (red) and DAPI (light blue) staining for the control and PlGF mouse heart sections. Scale bar = 50 μm. Please click here to view a larger version of this figure.
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 to perform immunostaining for cTnT on the same sections. Finally, we demonstrate how to merge the resulting color and fluorescent images. This protocol has been optimized for Formalin Fixed (4 °C, O/N)/Paraffin embedded mouse heart tissues, with the use of DIG-labeled LNA miRNA probes and cTnT. Further optimization may be required for different tissues, probe or antibody types, as outlined below.
RNase-free conditions should be carefully implemented throughout the steps that lead up to probe hybridization, as RNase contamination can severely compromise the outcome of the experiment. All solutions should be made with DEPC-treated ddH2O, and all Coplin jars should be treated with an RNase decontamination solution. Additionally, the following considerations should be taken into account when working with different DIG-labeled LNA miRNA probes. The hybridization temperature depends on the melting temperature (Tm) of each probe. As a general rule, a temperature that is 30 °C below the given RNA Tm or 20 °C below the given DNA Tm should be used for probe hybridization. Furthermore, the ideal probe concentration should be optimized. It usually lies between 1–50 nM. Lastly, the probe containing hybridization solution may be heated to 95 °C for 5 min to denature any secondary structures, if this is a concern. An important step to test the specificity of a probe, especially when used for the first time, is to include a negative (scrambled) as well as positive (for example U6) control probe, to ensure experimental success (Figure 2). Similar controls (for example a CD31 endothelial antibody) should be used during the immunostaining step.
A common problem in in situ hybridization experiments is the development of staining artifact/background signal. To avoid or minimize artifact staining steps, the tissue should be protected from drying out throughout all steps, specifically during long incubations. We recommend covering the slides with RNase-free coverslips during the hybridization step, particularly when high temperatures are used, and the use of humidifying chambers. We also suggest the use of a 568 or 594 fluorophore to detect cTnT or the protein of interest during the immunostaining step. This will eliminate any autofluorescence that often arises with the use of 488 fluorophores used on formaldehyde fixed tissues.
Another variable we recommend paying attention to is the duration of AP staining development, which depends on the levels of the specific miRNA present in the tissue. We suggest checking AP staining progression every 2 h, for the first experiment. Further optimization may include changing the temperature of incubation (RT-37 °C) parameter. Lastly, if background begins to develop before the true miRNA probe staining, or if the AP staining solution changes color to blue-brown, we suggest washing the slides in PBST twice, and replacing the staining solution with a freshly made one.
Finally, although this protocol has been optimized for Formalin-Fixed/Paraffin-embedded mouse heart tissues, it may be adapted for alternatively processed tissues (PFA fixed/OCT embedded) and/or the use of the different probe or antibody types. A limitation of combining in situ hybridization and immunostaining that should be considered is the failure of several antibodies to bind to their respective epitopes, due to the possible destruction of the latter ones at the high temperatures used during the hybridization and stringency washing steps. Further limitations of both in situ hybridization and immunostaining techniques most often refer to the power of these techniques to detect very low concentrations of miRNA or protein molecules respectively. Signal amplification kits are available to use for either DIG or antibody detection when miRNA or protein abundance is a concern. Such kits also provide a fluorescence alternative for the detection of miRNAs, which, when coupled with fluorescent immunostaining can simplify image acquisition. Alternative methods, such as Real-Time PCR and Western Blotting that have greater sensitivity, can also address low abundance issues. However, contrary to in situ hybridization/immunostaining protocol, these techniques provide no information on the tissue-specific localization of the miRNA/protein molecules.
To summarize, we describe a protocol that provides simultaneous detection of miRNA and protein molecules on the same tissue, which we believe will be an invaluable tool in miRNA research on mouse models.
The authors have nothing to disclose.
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).
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 |