Superscript 3 first strand synthesis system for rt pcr

Isolation of total RNA was performed using TRIsure (Nippon Gene, Toyama, Japan) according to the manufacturer's protocol after 48 h of the transfection. Total RNA (2 μg) was reverse transcribed into cDNA using Superscript III First-Strand Synthesis System for RT-PCR (Takara Bio Inc.) following the supplied protocol. The PCR reaction was performed using the KAPA SYBR Green Master Mix (5 μl KAPA SYBR Green Master Mix, 0.3 μl each of 10 μM primers, and 4.7 μl cDNA). PCR was performed using 7900 Fast Real-Time RT-PCR System (Applied Biosystems, Foster City, CA, USA) under the following condition: 42 cycles of two-step PCR (95 °C for 2 sec, 60 °C for 1 min). Forward primer—5′-CAATTGAGCGGACAACAGAA-3′ and reverse primer—5′-TTGGAAGGTCAGGGATATTAGC-3′ were used to amplify cDNA of the PALMD. Gene expressions were normalized with GAPDH.