**5.1. Sample reaction: (for the prestin gene promoter region in guinea pigs)**

Methylation-specific polymerase chain reaction: DNA purity was measured at wavelengths of 260 and 280 nm on spectrophotometry, and DNA quantification was performed using the DNA (μg/mL) = A260 × Dilution Factor × 50 (coefficient) formula at 260 nm UV. Subsequently, 10 μL of DNA was taken from each sample and bisulphite modification was carried out for DNA with Millipore CpGenome modification kit according to the manufacturer's instructions. This modification converts the cytosines of the unmethylated region to thymines. For the region thought to be altered in this manner, CpG sequences in exon 1 of the prestin gene were detected using the MethPrimer V1.1 beta program [30]. MSP was conducted according to the following protocols to investigate methylation utilising the PCR primers for exon 1 of the prestin gene stated below, and PCR conditions for the methylated and unmethylated regions are as follows:
