Fam reporter dye

Extracted RNAs for IL-6R mRNA assessment were subjected to reverse transcription using qScript cDNA SuperMix™ (Quantabio, Beverly, MA). Complementary DNAs (cDNAs) were subjected to quantitative real-time RT-PCR using PerfeCTa SYBR™ Green FastMix (Quantabio, Beverly, MA). Meanwhile, miRNAs were assessed by subjecting them to reverse transcription using the TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). cDNAs were subjected to quantitative real-time RT-PCR analysis using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA). All PCR reactions were performed in 96-well plates using the StepOnePlus™ real-time PCR System (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control during mRNA PCR, whereas SNORD38B labeled with FAM reporter dye (Applied Biosystems) was used as an endogenous control during miRNA PCR. In the experiments, parental cells or negative control were set as the reference. miR-34a expression was quantified using the comparative method (2^−ΔΔCT), where CT = threshold cycle, ΔΔ CT = (CT_miR-34a − CT _SNORD38B) − (CT _reference − CT _SNORD38B).

Utricles and saccules were collected separately from five WT mice at postnatal time points: P2, P14, P28, P60. RNA extraction and purification utilized the RNeasy Mini Kit (Qiagen). Total RNA was measured by spectrophotometer (Nanodrop, ND100, Thermo Fisher Scientific) and reverse transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR reactions were then performed with the TaqMan Gene Expression Assays with intron spanning Tmc1 (5′-CATCTGCAGCCAACTTTGGTGTGT-3′ and 5′-AGAGGTAGCCGGAAATTCAGCCAT-3′) and Tmc2 (5′-AGATCTTTGCGTTCCTTGCCAACC-3′ and 5′-GATCTTCTTTCGCAGCTGGGCATT-3′). TaqMan probes were labeled with FAM reporter dye (Applied Biosystems). Cycle threshold (Ct) values of triplicate reactions were measured for each sample. Tmc1 and Tmc2 plasmid DNA of known concentrations and lengths (1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000) were used to validate Tmc1 and Tmc2 primer efficiency and to estimate particle number across all time points.