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

Methods Mol Biol. 2019;1914:169-196. doi: 10.1007/978-1-4939-8997-3_9.

Abstract

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method 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 .

Keywords: Bone; Bone sarcoma; Chromogenic in situ hybridization; DNA; Gene expression; Histology; LNA probes; Primers; RT-qPCR; mRNA; miRNA.

MeSH terms

  • Animals
  • Bone and Bones / cytology*
  • Cell Line, Tumor
  • DNA / genetics
  • DNA / isolation & purification
  • Digoxigenin / chemistry
  • Gene Expression Profiling / instrumentation
  • Gene Expression Profiling / methods
  • Histocytological Preparation Techniques / instrumentation
  • Histocytological Preparation Techniques / methods
  • Humans
  • In Situ Hybridization / instrumentation
  • In Situ Hybridization / methods*
  • Mice
  • Mice, Nude
  • MicroRNAs / genetics
  • MicroRNAs / isolation & purification
  • Oligonucleotides / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • Real-Time Polymerase Chain Reaction / instrumentation
  • Real-Time Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / instrumentation
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Xenograft Model Antitumor Assays

Substances

  • MicroRNAs
  • Oligonucleotides
  • RNA, Messenger
  • locked nucleic acid
  • DNA
  • Digoxigenin