Sybr premix dimereraser perfect real time kit

Total RNA was isolated from cultured cells by TRIzol reagent (Invitrogen, Grand Island, NY, USA) Reverse transcription was performed by PrimeScript RT reagent Kit With gDNA Eraser (TaKaRa), and the quantitative RT-PCR was performed using SYBR Premix DimerEraser (Perfect Real Time) kit (TaKaRa) on the Roche LightCycler480 (Roche, San Francisco, CA, USA). Data were analyzed by the -2^ΔΔct method and the expression of β-actin was used as normalization control.

Cells were lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was detected by a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China) and separated by SDS-PAGE (Gene Molecular Biotech Inc, Shanghai, China). The primary antibodies were used: anti-FAM83D (1:1000, Biorbyt), GAPDH, Vimentin, E-cadherin, and N-cadherin (1:1000, CST). Anti-GAPDH antibody was used as the loading control.
Total RNA was extracted by TRIzol reagent (Invitrogen, Calsbad, CA) and reversely transcribed using the ReverTraAceqPCR RT kit (Toyobo, Shanghai, China). The qRT-PCR was performed using SYBR Premix DimerEraser (Perfect Real Time) kit (TaKaRa Bio Inc) on an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, USA) under the following conditions: 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. Data were analyzed using the -2ΔΔct method and the expression of GAPDH was used as normalization control. All primer sequences used are summarized in Table S1.

Total RNA extraction was isolated using TRIzol reagent (Ambion, Austin, TX, USA). The reverse transcription was performed with a 5 × PrimeScript RT Master Mix for Real-Time kit (TaKaRa, Tokyo, Japan). qRT-PCR was done using an SYBR® Premix DimerEraser™ Perfect Real-Time kit (TaKaRa). All operating procedures followed the supplier’s instructions. RT-qPCR analyses were done with a Mastercycler (Eppendorf, Hamburg, Germany). The primer sequences were as follow: TGM2, F: 5′- CAG CAG GGC TTT ATC TAC CA-3′, R: 5′- GAT CCC ATC TTC AAA CTG CC-3′; LDHA, F: 5′- TTC CAG TGT GCC TGT ATG G-3′ R: 5′- TTA TCA GTC CCT AAA TCT GGG TG-3′; LDHB, F: 5′- ACA ATA AGA TCA CTG TAG TGG G-3′, R: 5′- CAT CAG CCA GAG ACT TTC C-3′; GAPDH, F: 5′-GAA GGC TGG GGC TCA TTT GCA GGG-3′, R: 5′-GGT GCA GGA GGC ATT GCT GAT GAT-3′. The relative fold-change was normalized to GAPDH and calculated by the 2^-ΔΔCt method.