RNA Extraction and Expression Analysis

The TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA, including microRNA, according to the manufacturer’s instructions. For mRNA expression, cDNA synthesis was performed with Oligo (dT), and a SYBR RT-PCR kit (Takara, Otsu, Japan) was used for mRNA quantification with specific primers. For the detection of Cdr1as expression, specific primers were used (Forward: 5’-GTGTCTCCAGTGTATCGGCG-3’; Reverse: 5’-TACTGGCACCACTGGAAACC-3’) as described before [26 (link)]. For miR-7a expression, microRNA was analyzed by using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) with provided RT-U6 and microRNA-specific stem-loop primers, and the expression levels were determined through TaqMan MicroRNA assays with the TaqMan Universal PCR Master Mix (Applied Biosystems). All reactions were preceded on the ABI 7500 real-time PCR system (Applied Biosystems) by standard protocols. The comparative threshold cycle (Ct) value was calculated and analyzed by using the 2^-ΔΔCT method, with U6 and β-actin as an internal control.