Real-time PCR of PERK, eIF2α, ATF4, and CHOP in PBMCs

To determine the expression status of the PERK-CHOP pathway in patients with SSNHL, we examined the messenger RNA (mRNA) levels of PERK, eIF2α, ATF4, and CHOP in PBMCs of patients with SSNHL before treatment. Total RNA was extracted from the PBMCs using TRIzol reagent. Complementary DNA was then obtained by reverse transcription. Real-time polymerase chain reaction (rt-PCR) was performed with the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Singapore). The thermal cycle conditions for rt-PCR included an initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 5-s denaturation at 95°C and a 30-s extension at 60°C. The mRNA levels were normalized to the levels of the internal reference standard, β-actin, and determined by using the 2^−ΔΔCt method [12]. Each experiment was repeated 3 times. Table 1 lists the primer sequences of PERK, eIF2α, ATF4, CHOP, and β-actin that were used in rt-PCR.

Primer sequences for PERK, eIF2α, ATF4, CHOP, and β-actin used in rt-PCR.