Tissue Harvest and RNA extraction
+ +The animals were sacrificed under saturated isoflurane. Brains from the animals were dissected under a cold plate (≤4o C), and hippocampus were weighted and snap frozen in dry ice bath with isopentane and stored at −80°C until RNA extraction.
+ +RNA Extraction
+ +Total RNA was extracted using Direct-zol RNA Miniprep Plus Kits (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Approximately 15 mg of hippocampus tissue was added into a 2 ml tube containing 1 ml TRI Reagent® and one 5 mm stainless steel bead (Qiagen, Hilden, Germany). The tissue was homogenized for 2 min in a Tissue Lyser II (Qiagen, Hilden, Germany) with a speed frequency of 30 rpm followed by incubating for 5 min. 0.1 ml of 1‑bromo-3‑chloropropane was add into the homogenate, shaken vigorously for 15 s, and centrifuged for 15 min at 12,000×g at 4 °C. 600 µl upper aqueous was then transferred into a new collection tube containing 600 µl 100% ethanol. The mixture was loaded into a Zymo-Spin IIC column, wash once with RNA Prep Buffer and twice with RNA Wash Buffer. All RNA had been treated with DNase to eliminate possible DNA contamination, and further precipitated with ethanol. RNA purity and integrity were verified by a nanodrop spectrophotometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
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