Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization

Methods Mol Biol. 2025:2885:209-246. doi: 10.1007/978-1-0716-4306-8_11.

Abstract

This chapter is a revised version of the first Edition which aims to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or bone tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and methods to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes or using RNAscope technology.

Keywords: Bone; Bone Sarcoma; Chromogenic in situ hybridization; DNA; Formalin-fixed EDTA decalcified and paraffin-embedded tissues; Gene expression; LNA probes; Multiplex fluorescent assay; Primers; RNAscope; RT-qPCR; mRNA; miRNA.

MeSH terms

  • Animals
  • Bone and Bones* / cytology
  • Bone and Bones* / metabolism
  • DNA* / analysis
  • DNA* / genetics
  • In Situ Hybridization* / methods
  • Mice
  • MicroRNAs* / genetics
  • Paraffin Embedding
  • RNA, Messenger* / genetics
  • Real-Time Polymerase Chain Reaction* / methods

Substances

  • MicroRNAs
  • RNA, Messenger
  • DNA