Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNAs were extracted using TRIzol® Reagent (Invitrogen) and reverse transcribed with High Capacity cDNA Reverse Transcription kit (Applied Biosystems) for mRNAs or with Mir-X^™ miRNA First-Strand Synthesis Kit (Clontech) for miRNAs. Quantitative PCR was performed with Power SYBR Green PCR Master Mix, ABI 7500, and ABI 7900 (Applied Biosystems) using default thermal cycling program. Except U6 Primer and universal reverse primer (Clontech), all other primers were purchased at Sigma Life Science (Sigma). Endogenous Gapdh, U6, or sno202 was used as normalization controls for mRNAs and miRNAs, as indicated. Relative levels of mRNAs and miRNAs were calculated by comparative Ct method using ABI 7500 software (version2.0.5) and GeneEx 5.3.2 (Multid analyses). The primer sequences used were: Bace1, 5′-CAGTGGGACCACCAACCTTC-3′ and 5′-GCTGCCTTGATGGACTTGAC-3′; Gapdh, 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′. miR-186 mature sequence and universal reverse primer were used for miR-186.