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

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.

Figure 1. Callus Extract Protein Yields and Quality

Panel A: Soluble protein levels were measure in callus extracts after 1 hour or 24 hours of dialysis to remove RNAlater. Samples sizes were LN-MPER = 12 (not dialyzed); LN-RL 1 hour = 8; RL-MPER 1 hour = 4; RL-RL 1 hour = 4; LN-RL 24 hour = 3; RL-MPER 24 hours = 3; RL-RL 24 hours = 2. Error bars represent standard errors of the mean. Panel B: Aliquots of soluble protein from extracts prepared using each method were separated by SDS-PAGE and detected by Coomassie Blue staining. Positions for protein standards are shown.

Figure 2. Variation in Cytokine Recovery using Different Extraction Methods

Selected cytokine protein levels from LN-MPER, LN-RL, RL-MPER, and RL-RL samples were normalized against the mean value for the LN-MPER samples as the percent change. These data are shown as mean values (± SEM). Significant differences were determined using One Way ANOVA or Kruskall Wallis tests. Post-hoc tests was used to determine the differences between groups. Dunnett post-hoc method was used when sample sizes were the same and Dunn’s post-hoc method was used when sample sizes were different in each group. Differences were considered to be significant at P < 0.05. The statistical analysis was performed using SigmaPlot 12.5 software (Systat Software Inc.).

Figure 3. Representative Chromatograms from LC-MS/MS Measurements

Lipids extracted from a mouse femur fracture callus frozen in liquid nitrogen and pulverized in RNAlater were derivatized and then separated on a reverse phase column. Endogenous eicosanoids (black lines) and deuterated internal standards (gray lines) were detected by MS/MS. Shown are chromatograms for TXB² (A), PGD² (B), and PGE² (C) from a single fracture callus.