Transscript all in one first strand cdna synthesis supermix for qpcr one step gdna removal kit

The expression levels of atrogin 1 and MU-RF1 mRNA in gastrocnemius were detected by RT-qPCR. The right gastrocnemius tissues were lysed in 1 ml TRIzol to extract the total RNA according to the manufacturer's instructions, and RT was performed. The atrogin 1, MU-RF1 and GAPDH primers details and products fragments as listed in Table I. cDNA was synthesized using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (Beijing TransGen Biotech Co., Ltd., Beijing, China), the concs were 4 µl total RNA; 1.6 µl 5X TransScript All-in-One SuperMix for qPCR; 0.4 µl gDNA Remover; and 2 µl RNase-free water, under the conditions of 37°C for 15 min and 85°C for 5 sec. Subsequently, fluorescence qPCR was performed with reactions consisting of 18.5 µl H₂O, 2.5 µl 10X SYBR green PCR buffer (SYBR^® Fast qPCR Mix; Takara Biotechnology Co., Ltd., Dalian, China), 0.5 µl dNTPs, 0.5 µl Taq, 0.5 µl forward primer (10 pmol/µl), 0.5 µl reverse primer (10 pmol/µl) and 2 µl cDNA, and incubation at 93°C for 2 min, followed by 40 cycles of 93°C for 15 sec, 55°C for 25 sec and 72°C for 25 sec. Finally, the cycle threshold (Cq) values were calculated to analyze the relative expression level (ΔCq=Cq_{target gene}−Cq_{reference gene}; ΔΔCq=ΔCq−ΔCq_max) (9 (link)).