Sybr premix ex taqtm 2 pcr kit

Total RNA was extracted using the Trizol reagent (Invitrogen, USA) as per the instruction of the manufacturer’s manual. The extracted RNA was quantified using a NanoDrop device (ThermoFisher Scientific) and stored at − 80 °C pending subsequent analysis. The PrimeScript RT Reagent Kit (Takara, Japan) was used for reverse transcription of total RNA into complementary DNA (cDNA). The expression level of MCT1 and MCT4 were assessed by RT-qPCR using the SYBR^®premix ExTaqTM II PCR Kit (Takara, Japan). The primers were synthesized by Sangon, Shanghai, China, and the sequences are as follows:

MCT1, Forward, 5′-TGGATGGAGAGGAAGCTTTCTAAT-3′.

Reverse, 5′-CACACCAGATTTTCCAGCTTTC-3′.

MCT4, Forward, 5′-CACGGCATCGTCACCAACT-3′.

Reverse, 5′-ACAGCCTGGATAGCAACGTACAT-3′.

GAPDH, Forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′.

Reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′.

GAPDH was the housekeeping gene used for the control. The experiments were performed 3 times and triplicated wells were used in each group in one experiment. The reaction conditions for PCR were set as follows: pre-denaturation at 95 °C for 30 s, 45 cycles of denaturation at 95 °C for 5 s, and annealing at 58 °C for 34 s. The 7500 Real-Time PCR System (Thermo Fisher Scientific) was used to perform the assay. The 2^−ΔΔCt method was used to calculate the expression of the target gene between the experimental group and the control group.

RNA was reverse transcribed into cDNA using the PrimeScript RT Master kit (Takara, RR037A). RT-qPCR was performed with the SYBR® Premix EX TaqTM II PCR Kit (Takara, DRR041A) following the manufacturer’s instructions on the Roche Lightcycler 480 Real-Time PCR System. Data were calculated according to the Applied Biosystems comparative Ct method. Primers used for LncRNA NEAT1 and GAPDH qRT-PCR were as follows:
LncRNA NEAT1-Forward: 5′-GGA​TGA​GGC​CTG​GTC​TTG​T-3’.
LncRNA NEAT1-Reverse: 5′-GAG​AAA​AGT​CCA​AAA​GGA​GCA​C-3’.
GAPDH-Forward: 5′-ACA​ACT​TTG​GTA​TCG​TGG​AAG​G-3’.
GAPDH-Reverse:5′-GCCATCACGCCACAGTTTC-3’.

Total RNA was extracted with TRIzol reagent (Invitrogen) and then reverse transcribed to cDNA according to the protocol of the miRcute miRNA First-strand cDNA synthesis kit (Beijing Tiangen Biotechnology, Ltd). cDNA was subjected to real-time PCR using a SYBR premix Ex TaqTM II PCR kit (Takara, Dalian, Liaoning Province, China) with the ABI 7500 real-time PCR instrument. Quantitation of miR-519d was normalized to U6, and LEP was normalized to GAPDH. The primer sequences are listed in Table S1. Results were calculated by 2^−ΔΔCT method.

The total RNA from the cultivated cells was extracted with Trizol reagent (Invitrogen, California, USA) in accordance with the instructions provided by the manufacturer. In the second step, 2 μg of total RNA was subjected to reverse transcription utilizing a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, Niaoning, China) in an overall volume of 20 L in accordance with the procedure provided by the manufacturer. After that, a real-time quantitative polymerase chain reaction (qRT-PCR) was performed with the SYBR Premix Ex TaqTM II PCR Kit (Takara, Otsu, Shiga, Japan) by carefully adhering to the instrument that was provided by the manufacturer. Results were normalized to the content of GAPDH. The results of each experiment were recorded three times. The 2^−ΔΔCt method was used to represent the fold-change values of lncRNA expression. Primers used for RT-RCR are presented as follows: SSTR5-AS1-F: 5′-ACTACAGGTGCCATCAGACC-3′, SSTR5-AS1-R: 5′-AGCCTGCCATCCTAACACTT-3′; GAPDH-F: F: 5′-AGGTGAAGGTCGGAGTCAACG-3′, GAPDH-R: R: 5′-AGGGGTCATTGATGGCAACA-3′.

Total RNA from WT and SCARKO mice was prepared with Trizol reagent according to the manufacturer’s instructions. Then RNA was reverse transcribed into cDNA using the PrimeScript RT Master kit (Takara, RR037A). RT-qPCR was performed with the SYBR^® Premix EX Taq^TM II PCR Kit (Takara, DRR041A) following the manufacturer’s instructions on the Roche Lightcycler 480 Real-Time PCR System. Data were calculated according to the Applied Biosystems comparative Ct method.