Mircury lna microrna array kit

For RT-qPCR analysis, total RNA was extracted from hippocampus tissues 7 days after anesthesia using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Then, 2 µg of total RNA was used as a template to synthesize the first-strand cDNA using iScriptTM Reverse Transcription Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the mRNA and miRNA expression levels were determined using a Ssofast EvaGreen Supermix kit (Bio-Rad Laboratories, Inc.) with an ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a miRCURY LNA microRNA Array kit (version 14.0; Exiqon; Qiagen, Hilden, Germany). The primers (Table I) were synthesized by Shanghai Sangon Biologic Engineering Technology and Services Co., Ltd. (Shanghai, China). GAPDH was used as the internal control for mRNA quantifications and U6 was used as the internal control for miRNA quantification. The reaction conditions were as follows: 95°C 10 min, followed by 95°C for 30 sec, 60°C for 40 sec and 72°C for 10 sec for 40 cycles. All reactions were run in triplicate. The relative mRNA expression level was calculated by the 2^−ΔΔCq method (27 (link)).