Abstract
MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding the in vivo localization of miRNAs is an important step for uncovering their roles in brain function. However, the in situ detection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization of in situ hybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37°C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 × standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqManTM primers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in the in vivo localization of miRNAs, and the elucidation of the specific roles of miRNAs.
Highlights
MicroRNAs serve as post-transcriptional fine-tuners of gene expression (Meister, 2007), and have become a focus of interest in both basic and translational neuroscience research (Soreq, 2014)
Neuron-specific miR-124a is useful for optimizing the detection sensitivity and performance of the miRNA in situ hybridization (ISH) method in brain tissues, since miR-124a is expressed in abundance in the central nervous system (Deo et al, 2006)
We performed TaqManTM RT-PCR and showed that the relative expression levels of miR-181a and miR-34a in the hippocampus were one-half and one-eighth of those of miR124a, respectively. These data indicate that fine-tuning of the miRNA ISH protocol is required, especially for the detection of low-abundance miRNAs
Summary
MicroRNAs (miRNAs) serve as post-transcriptional fine-tuners of gene expression (Meister, 2007), and have become a focus of interest in both basic and translational neuroscience research (Soreq, 2014). The present study was designed to investigate the effects of a series of critical procedures, including the following: (1) tissue fixation, (2) proteinase K treatment, (3) hybridization temperature, and (4) stringent washes Using this optimized protocol, we detected the ISH signals specific to several miRNAs, whose expression patterns are unknown. 1. 10× phosphate buffered saline (PBS), pH 7.38 30 g of NaCl. 1 g of KCl. 12 g of Na2HPO412H2O. 5. 10× Tris-HCl, NaCl (TN) buffer, pH7.3 13.4 g of Trizma base. 7. 1 M Tris-HCl, pH 9.5 Dissolve 121 g of Trizma base in 900 mL of ultrapure water. Alkaline phosphatase reaction buffer 100 mL of 1 mM Tris-HCl, pH 9.5. Biotin amplification reagent working solution Dilute the biotin amplification reagent stock solution from the TSA Plus biotin kit 1:50 with the 1× plus amplification diluent from the same kit (PerkinElmer)
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