Method for measuring lipid mediators, proteins, and messenger RNAs from a single tissue specimen

Anal Biochem. 2015 Jan 15:469:34-42. doi: 10.1016/j.ab.2014.10.004. Epub 2014 Oct 20.

Abstract

This article describes a new method for extracting RNA, protein, and lipid mediators from a single tissue specimen. Specifically, mouse bone fracture callus specimens were extracted into a single solution that was processed using three different procedures to measure messenger RNA (mRNA) levels by reverse transcription-quantitative polymerase chain reaction (RTqPCR), cytokines and growth factors using an xMAP method, and lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method has several advantages because it decreases the number of animals necessary for experimentation, allows division of the sample from a homogeneous mixture that reduces sample variability, and uses a solution that protects the integrity of the macromolecules during storage.

Keywords: Bone regeneration; Fracture healing; Lipid mediator; Metabolomics.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bony Callus / metabolism
  • Bony Callus / pathology
  • Chromatography, High Pressure Liquid
  • Cytokines / analysis
  • Fractures, Bone / metabolism*
  • Fractures, Bone / pathology
  • Intercellular Signaling Peptides and Proteins / analysis*
  • Lipids / analysis*
  • Lipids / isolation & purification
  • Mice
  • RNA, Messenger / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Solid Phase Extraction
  • Tandem Mass Spectrometry

Substances

  • Cytokines
  • Intercellular Signaling Peptides and Proteins
  • Lipids
  • RNA, Messenger