Stereotaxic injection of siRNA was performed as described previously [22 (link), 32 (link)]. In brief, mice were anesthetized with a mixture (3.5:1) of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (23.3 mg/ml) at a dose of 2.5 μl/g body weight. One portion of diluted siRNA (50 ng/μl) was mixed with 2.5 portions Neurofect transfection reagent (T800075, Genlantis, San Diego, CA, USA), and 0.5 portion of 50% sucrose 20 min prior to injection and incubated on ice.
The siRNA mix (each, 1.8 μl of 7.5 ng/μl) was injected into the CA3 region (stereotaxic coordinate: AP, -1.9; ML, ±3.0; DV, -2.1 mm) on both sides using a stereotaxic injection system (Vernier Stereotaxic Instrument, Leica Biosystems, Wentzler, Germany) and a Hamilton syringe with a 30-G needle. After 5 min, the needle was removed in three intermediate steps for 3 min each. Mice were kept on a warm pad until awakened. Tissue samples with siRNA injection were prepared 48 h after injection.
Control siRNA (siCON, SN-1012) and MeCP2-siRNA (siMeCP2, 1385135; NM_001081979.2) were purchased from Bioneer Co. (Daejeon, Korea). The siRNAs were resolved to 50 ng/μl in a siRNA dilution buffer (B-002000-UB-100, Dharmacon, Lafayette, CO, USA).
The siRNA mix (each, 1.8 μl of 7.5 ng/μl) was injected into the CA3 region (stereotaxic coordinate: AP, -1.9; ML, ±3.0; DV, -2.1 mm) on both sides using a stereotaxic injection system (Vernier Stereotaxic Instrument, Leica Biosystems, Wentzler, Germany) and a Hamilton syringe with a 30-G needle. After 5 min, the needle was removed in three intermediate steps for 3 min each. Mice were kept on a warm pad until awakened. Tissue samples with siRNA injection were prepared 48 h after injection.
Control siRNA (siCON, SN-1012) and MeCP2-siRNA (siMeCP2, 1385135; NM_001081979.2) were purchased from Bioneer Co. (Daejeon, Korea). The siRNAs were resolved to 50 ng/μl in a siRNA dilution buffer (B-002000-UB-100, Dharmacon, Lafayette, CO, USA).
