Mirneasy mini kit with dnase digestion

Sample tissues were placed in RNAlater solution (Life Technologies) and stored at −80 °C until further processing. Specimens were then thawed on ice and placed in Qiazol lysis reagent (Qiagen). Tissue was homogenized using the Tissue Tearor (BioSpec Products) and mRNA was extracted using the miRNeasy Mini Kit with DNase digestion (Qiagen) to remove DNA contamination. RNA yield was determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific). One µg of mRNA was reverse transcribed in 20 µL of cDNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems). Gene expression was quantified using a 7500 Fast Real-Time PCR system (Applied Biosystems) from a total of 10 µL of Master Mix per well, which included 1 × Fast SYBR Green (Applied Biosystems), forward and reverse primers (0.45 µM), and 0.5 µL of cDNA. For each gene of interest, samples were run in duplicate and control wells were run to rule out DNA contamination and primer dimer binding. Proper amplicon production was confirmed by melt curve analysis. Data were normalized to the housekeeping gene, β-actin, and presented as fold change expression to WT whole bone, calculated using the formula 2^−ΔΔC(t). β-actin C(t) values were stable across treatment groups.