Sybr primescript rt pcr kit

The total RNA of cells was isolated using TRIzol Reagent (Invitrogen, CA, USA). The total RNA of exosomes was extracted using TRIzol Reagent (Invitrogen, CA, USA) and Dr. GenTLE™ Precipitation Carrier (Takara, Dalian, China). Then, 1 μg total RNA was added to a mixed reagent for reverse transcription. Reverse transcription for miRNA was performed according to the manufacturer's instructions for the RevertAid First Strand cDNA Synthesis Kit (Invitrogen, CA, USA) or Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Dalian, China). For mRNA, Total RNA was subjected to reverse transcription using a PrimeScript RT Reagent kit (Takara, Dalian, China) following the manufacturer's protocol.
The expression levels of miRNA and mRNA were measured by real-time PCR using SYBR Prime Script RT-PCR Kit on a LightCycler^® 480 system (Roche, Switzerland) following the manufacturer's protocol. In brief, the reactions were incubated at 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The relative expression levels of miRNA and mRNA were quantified by the 2^-ΔΔCT method and normalized using U6 or Actin expression. The primers are shown in Table S1.

Total RNA was extracted using RNAzol (Molecular Research Center, Inc, USA) agent. It was dissolved in DNase/RNase-free water (Ribobio, Guangzhou, China) and quantified using NanoDrop2000. RNA reverse transcription process was performed following the manufacturer’s instructions (PrimeScript RT reagent Kit, Takara, Japan). RT-PCR was performed using SYBR PrimeScript RT-PCR Kit (Roche, USA) following the manufacturer’s protocol. Each group was duplicated three times in one experiment.
Primer sequences:
MLKL: Forward, 5′- CAACCTGAAGTAACAGCGAGA-3′
Reverse, 3′-GGCTAATGGGGAGATAGAAAA-5′
GAPDH: Forward, 5′-CCCTGTTGCTGTAGCCAAATT-3′
Reverse, 3′-CACCCACTCCTCTACCTTCGA-5′

Real-time quantitative RT-PCR was performed to assess the mRNA expression levels of TNF-α, IL-1β, IL-6, and IL-10 in AR42J cells. The total RNA from the cells was extracted using an RNA Extract Kit and converted to first-strand cDNA according to the manufacturer’s instructions. Quantitative real-time PCR was performed with an SYBR PrimeScript^TM RT-PCR Kit and a LightCycler System (Roche Diagnostics, Lewes, UK). The primer sequences used for PCR were as follows: TNF-α sense 5’-CCAGGAGAAAGTCAGCCTCCT-3’ and antisense 5’-TCATACCAGGGCTTGAGCTCA-3’, resulting in an 87-bp product; IL-1β sense 5’-CACCTCTCAAGCAGAGCACAG-3’ and antisense 5’-GGGTTCCATGGTGAAGTCAAC-3’, resulting in a 79-bp product; IL-6 sense 5’-CTGGTCTTCTGGAGTTCCGTTTC-3’ and antisense 5’-CATAGCACACTAGGTTTGCCGAG-3’, resulting in a 301-bp product; IL-10 sense 5’-GGCTCAGCACTGCTATGTTGCC-3’ and antisense 5’-AGCATGTGGGTCTGGCTGACTG-3’, resulting in a 116-bp product; and β-actin sense 5’-GAACACGGCATTGTAACCAACTGG-3’ and antisense 5’-GGCCACACGCAGCTCATTGTA-3’, resulting in a 77-bp product. Amplification was performed under the following conditions: 95°C for 30 s followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 20 s [17 (link)]. All reactions were performed in triplicate.