Neonatal T3 Exposure Alters Cochlear Gene Expression

Neonatal mice were injected with T3 at P0 and P1, then sacrificed at P4. The cochleae were removed and dissected in cold Hanks’ balanced salt solution (H1045, Solarbio, beijing, China). The membranous cochlear duct sourced from one cochlea was used to generate one sample. The detailed methods for the RNA extraction and reverse transcription were as described previously [56 (link)]. Total RNA was extracted from the collected tissues using an RNAprep Pure Tissue Kit (Tiangen Biotech Co., Ltd., Beijing, China) and was reverse transcribed using a PrimeScript RT Reagent Kit with gDNA eraser (Takara Bio Inc. Shiga, Japan). RT-qPCR was performed in a Roche LightCycler 480 instrument (Roche Diagnostics Ltd., Basel, Switzerland). Real-time qPCR conditions were an initial denaturing step of 15 s at 95 °C followed by 40 cycles of 15 s denaturation at 95 °C, 60 s annealing at 60 °C, and 20 s extension at 72 °C. The transcriptional expression was normalized to the expression of GAPDH and the relative expression level between the control and T3 group was calculated using the 2^−∆∆CT method [41 (link)]. The real-time qPCR primers are shown in Supplementary Table S1.