Quantifying Cilium and Hh Signaling Genes

The supraspinatus tendon and enthesis were harvested, frozen in liquid nitrogen, and pulverized in a ball mill homogenizer (Mikro-Dismembrator U, Sartorius). Total RNA was extracted by TRIzol reagent and Invitrogen PureLink RNA Mini kit, as described in our previous study (2 (link)). The High-Capacity cDNA Reverse Transcription Kit (Invitrogen) was used to reverse transcribe RNA into cDNA. The relative abundance of cilium- and Hh signaling–related genes was evaluated by SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-PCR) on an Applied Biosystems QuantStudio 6 flex system. The sequences of RT-PCR primers used here are listed in table S1. Glyceraldehyde 3-phosphate dehydrogenase serves as housekeeping gene, and the relative mRNA expression level for each target gene was determined as 2^−ΔΔCt.