Total RNA were extracted from stages 3–6 (100 mg each) using TRIzol_Reagent with the PureLink_RNA Mini Kit (Ambion by Life Technologies Carlsbad, CA, USA). RNase-free mortars and pestles were used in combination with liquid nitrogen to disrupt frozen tissue samples into a powder. One hundred milligrams of powdered tissue samples in 1 mL TRIzol Reagent were homogenized by using a mortar and pestle26, and then through three cycles of pestle strokes at 200 riv/min in a potter Helvehjem homogenizer. Samples were centrifuged for 10 min at 12,000 g and the supernatant was removed and incubated for 3 min at room temperature. Then 0.2 mL chloroform per 1 mL TRIzol Reagent used were added, the tubes were vigorously shaken by hand for 15 s, incubated at room temperature for 3 min, and were centrifuged at 12,000 g for 15 min at 4 °C. The upper colorless phase containing the RNA was transferred to a new Rnase-free tube, adding an equal volume 100% ethanol and vortexing to mix well. Binding, washing, and elution steps were performed using the PureLink_RNA Mini Kit according to the manufacturer’s instructions (Ambion by Life Technologies Carlsbad, CA, USA). Then, the RNA was treated with Recombinant Rnase-free Dnase I from bovine pancreas (Roche Diagnostics, Indianapolis, USA) according to the manufacturer’s instructions. Absence of DNA was checked by comparing cDNA samples with RNA samples which were not reverse transcribed (minus RT control). The purity of all RNA samples was assessed at absorbance ratios of A260/A280 and A260/A230 using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, WA) and the integrity of the RNA was immediately checked using 1.2% agarose gel electrophoresis (1.3 µg samples). RNA concentration was calculated based on absorbance values at 260 nm, and RNA samples were stored at −80 °C until use.