Chloroform

The mixing of each PCL with the various concentrations of nHA followed the protocol outlined by Abdal-Hay et al. [15 (link)]. Briefly, PCL was dissolved in chloroform (Chem-Supply, Gillman, SA, Australia) at 15% w/v by vigorous stirring for 40 min. nHA was added in 10 mg batches with vigorous stirring for 5 min in between the batches. After all the nHA was added, the solution was ultrasonically agitated for 20 min followed by the vigorous stirring overnight. The next day, the solution was again ultrasonically agitated for 30 min before being cast in a petri dish and placed under vacuum for 3 days to allow the chloroform to fully evaporate.

Cell lysates were homogenized in 1 mL Trizol (Invitrogen, Waltham, MA, USA) and incubated at room temperature for 10 min. 0.2 mL chloroform (Chem Supply, Melbourne, Australia) per 1 mL Trizol was added to the samples and they were centrifuged at 12,000× g for 15 min at 4 °C. The colorless, aqueous phase of each sample containing the RNA was transferred into a fresh 1.7 mL microcentrifuge tube. RNA was precipitated by adding 0.5 mL Propan-2-ol (Chem Supply) per 1 mL Trizol and the samples were centrifuged again at 12,000× g for 10 min at 4 °C. The supernatant from the tubes was discarded, and the RNA pellet was washed with 75% ethanol (Chem Supply, Melbourne, Australia) in diethyl pyrocarbonate (DEPC)-treated water (Sigma, St. Louis, MO, USA), vortexed and centrifuged at 7500× g for 5 min at 4 °C. The RNA pellet was air-dried and redissolved in RNAse-free H₂O (Invitrogen, Waltham, MA, USA). The concentration and purity of the RNA samples was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Cells were lysed directly in the culture plates by the addition of 1 ml Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA). Lysate was transferred to microcentrifuge tubes and 200 μL of chloroform (Chem-supply, Gillman, SA, Australia) was added before centrifugation at 13,000 g at 4 °C for 10 min in accordance with the manufacturer’s instructions (ThermoFisher). The resultant aqueous phase, containing total RNA, was separated from the organic phase and mixed with 80 μg glycogen (Life Technologies, Mulgrave, VIC, Australia) and 500 μL isopropanol (Chem-supply, Gillman, SA, Australia), and incubated at −20 °C overnight for RNA to precipitate. After centrifuge at 9000 g at 4 °C for 30 min, the supernatant was discarded, and the pellet was washed with 75% cold ethanol twice. Finally, the RNA pellet was re-suspended in nuclease-free water and stored at −80 °C. The concentration and purity of extracted RNA was assessed by the Agilent small RNA kit and the 2100 Bioanalyzer according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). The provided system software was used for automatic calculation of RNA integrity number (RIN). All samples had RIN values above 8.5.