High capacity cdna reverse transcription kit with random hexamers

For each experimental condition, RNA (500 ng) was reverse transcribed using the high-capacity cDNA reverse transcription kit with random hexamers (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Tubulin was selected as the housekeeping gene using the Normfinder algorithm as the most stable reference among five candidates across all conditions. Primer sequences are reported in Supplemental Table S5. PCR was performed on a 7900 Fast Real-time PCR system (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA). Gene expression levels are plotted as 2^−∆ct, unless specified. Plots were generated using GraphPad Prism version 8.

Inner ears or the sensory epithelium of the cochlea and vestibule were dissected, frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted by the RNeasy Plus Mini Kit (Qiagen) and diluted in RNase-free water (Life Technologies). The Mouse Total RNA Tissue Panel (Clontech) was used to examine the presence and the level of the expression of lncRNAs in various tissues. cDNA was prepared from 1 ug of RNA using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems). mRNA expression was evaluated using the Fast SYBR® Green Master Mix (Applied Biosystems) in the StepOneTM Real-Time PCR System (Applied Biosystems).
Primers were designed for 80–150 base-pair (bp) segments using Primer3 (http://bioinfo.ut.ee/primer3/). All primers were validated, including the amplification efficiency and correlation coefficient (R²) of each primer pair; samples were examined using a standard curve with five cDNA dilutions. In addition, a melt curve was performed to verify the specificity of the primers. Primers are available upon request. No template control (NTC) samples were included as negative controls. The 2^−ΔΔCt method was used to calculate the expression of each lncRNA. mRNA expression was normalized to Gapdh. Cochlear tissue from postnatal day 0 was used as the control sample. The data in the figures are presented as the mean ± SD.