MicroRNAs (miRNAs) are key regulators of gene expression at the posttranscriptional level. Precisely profiling of miRNA expression will help us to better understand their roles in normal and diseased cells and tissues. Here we describe in situ miRNA detection by padlock probing and miRNA target-primed rolling circle amplification. We optimized our protocol and showed it can be applied to both fixed cells and tissue sections. The method can be used in basic research and potentially in clinical diagnostics in the future. (C) 2020 Elsevier Inc. All rights reserved.