One step reverse transcription kit

The rats were sacrificed by overdose of anesthesia, their fur was soaked in 70% ethanol, and they were decapitated. Auditory vesicles were removed in an ice bath to obtain the inner ears. Both ears of each animal were considered a metering unit. Total RNA was extracted. Reverse transcription-PCR was performed using a one-step reverse transcription kit (Qiagen, Dusseldorf, Germany), according to the manufacturer's instructions. β-Actin served as the internal reference. PCR conditions were as follows: reverse transcription at 50°C for 30 minutes; initial activation at 95°C for 15 minutes, 35 cycles of denaturation at 94°C for 50 seconds, annealing at 60°C for 50 seconds, extension at 72°C for 60 seconds; extension at 72°C for 10 minutes. XIAP primer sequences: upstream primer, 5′-AGG AAC CCT GCC ATG TAT TG-3′; downstream primer, 5′-TGT TGT TCC CAA GGG TCT TC-3′. β-Actin primer sequence: upstream primer, 5′-CGC ACC ACT GGC ATT GTC AT-3′; downstream primer, 5′-TIC TCC TTG ATG TCA CGC AC-3′. The product length was 494 bp. PCR products were electrophoresed on a 1.5% agarose gel. A Gel Imaging Analysis System (Tanon2020; Tanon, Shanghai, China) was employed to measure optical density. The expression of XIAP mRNA was calculated as the average optical density ratio of the target band to the internal reference band.