AUTHOR=Tsuneoka Yousuke , Funato Hiromasa 

TITLE=Modified in situ Hybridization Chain Reaction Using Short Hairpin DNAs

JOURNAL=Frontiers in Molecular Neuroscience

VOLUME=13

YEAR=2020

URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2020.00075

DOI=10.3389/fnmol.2020.00075

ISSN=1662-5099

ABSTRACT=<p>The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes. <italic>In situ</italic> hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although <italic>in situ</italic> HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional <italic>in situ</italic> hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified <italic>in situ</italic> HCR showed sufficient fluorescent signals to detect various mRNAs such as <italic>Penk</italic>, <italic>Oxtr</italic>, <italic>Vglut2</italic>, <italic>Drd1</italic>, <italic>Drd2</italic>, and <italic>Moxd1</italic> in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified <italic>in situ</italic> HCR in detecting the <italic>Oxtr</italic> mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified <italic>in situ</italic> HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified <italic>in situ</italic> HCR simultaneously detected the distributions of c-Fos immunoreactivity and <italic>Vglut2</italic> mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified <italic>in situ</italic> HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.</p>