Sarah Beck-Cormier, CR CNRS.
INSERM U791 - LIOAD - Laboratoire d’Ingénierie Ostéo-Articulaire et Dentaire. STEP group «Skeletal Tissue Engineering and Physiopa- thology». Nantes, France.

Overview

  • Keywords: Mouse bone samples, RNA extraction, FastPrep-24™ homogenizer, gene expression
  • Aim of the study: High quality RNA extraction from mouse bone samples
  • Application: qPCR
  • Sample Name: Mouse Calvaria, long bones, chondrocytes, Osteoblasts (OB).
  • Material: FastPrep-24™ homogenizer, Lysing Matrix S (metal beads), Lysing Matrix D (ceramic beads)
  • Buffer: RNA extraction buffer

Protocols & Parameters

  1. Put bone samples immediately in liquid nitrogen after
    sampling

  2. Place bone sample in Lysing Matrix tube containing 6 metal
    beads (Lysing Matrix S)

  3. Add 350 μl of RNA extraction buffer per sample

  4. Load tube in the FastPrep-24 ™ homogenizer and process
    2 x 15 sec at speed setting of 6m/s with 5 min intermediate incubation on ice

  5. Centrifuge Lysing Matrix tube at 12 000 x g, 5 min at 4°C

  6. Transfer supernatant to a new 2 ml microcentrifuge tube
    and follow RNA extraction according to RNA extraction
    protocol


Results

  • 1-4: 250-300 ng RNA from mineralized OB (Lysing Matrix D)
    5: 200 ng RNA from mineralized chondrocytes
    (Lysing Matrix S)

Conclusion

  • Bone sample grinding is challenging. This study shows that the use of FastPrep-24 ™ homogenizer in combination with metal beads
    is highly performant for this application.
  • High RNA quality and yield are extracted from mineralized Osteoblasts and Chondrocytes using FastPrep-24™, Lysing Matrix D
    & Lysing Matrix S tubes.
  • FastPrep® technology is the most adapted for bone & forensics studies.