Improved method for isolating high-quality RNA from mouse bone with RNAlater at room temperature

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Accurate gene expression analysis in bone requires high-quality RNA.
• RNA degrades rapidly upon thawing frozen bone tissue.
• Existing methods struggle to yield intact RNA from both marrow and mineralized bone.

Purpose of the Study:

• Develop simple, robust, and validated methods for high-quality RNA isolation from mouse femur shaft and marrow.
• Evaluate the efficacy of RNAlater for preserving RNA integrity in bone tissue.
• Compare RNA isolation methods for downstream gene expression analysis.

Main Methods:

• RNA isolation from mouse femur shaft and marrow using RNAlater preservation.
• Quantification of RNA integrity using the RNA Integrity Number (RIN) on a TapeStation.
• Comparison of TRI Reagent lysis versus a hybrid TRI Reagent/RNeasy column purification method.

Main Results:

• RNAlater significantly improved RNA quality, yielding mean RINs of 8.0 for femur shaft and 9.6 for femur marrow.
• High-quality RNA (mean RIN 9.3) was obtained from lumbar vertebral bone using RNAlater.
• RNAlater allowed thawing to room temperature before lysis without RNA degradation.
• The hybrid method yielded femur shaft RNA with fewer qRT-PCR inhibiting impurities compared to TRI Reagent alone.

Conclusions:

• RNAlater is an effective reagent for preserving RNA integrity in various bone tissues, including mineralized bone.
• The RNAlater preservation method simplifies sample handling by allowing room-temperature thawing.
• A hybrid RNA isolation method may be advantageous for reducing impurities for qRT-PCR analysis from mineralized bone.