The trachea from mice was carefully isolated, quickly removed of connective tissue and epithelium, and then frozen in dry ice. The total RNA of the trachea was isolated with the TRIZOL (Invitrogen) method according to the manufacturer’s guidelines, and cDNA was synthesized using extracted RNA with an Omniscript Reverse Transcription kit (QIAGEN). The specific primers for mouse RYR1, RYR2, RYR3, and for β-actin have been described previously (ZhuGe et al., 2006) and were synthesized by Invitrogen. β-Actin was used as a positive control and the absence of DNA as a negative control, and the PCR reaction was performed in a PCR mastercycler (model 5332; Eppendorf). The PCR was held at 94°C for 30 s and then cycled 35 times, with each cycle consisting of 93°C for 30 s, 54°C for 30 s, and 72°C for 30 s. The hippocampus for the same animal was dissected and checked for RYR mRNA with the same protocol as in ASM.
