Sybr primescript qpcr kit

Total RNA was extracted from the tissues or cells using the Trizol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The RNA samples were reverse-transcribed into cDNA with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Takara, Otsu, Japan). β-Actin was used as the internal control. All primers used for qPCR were obtained from GenePharma (Shanghai, China), and the sequences are listed in Table 2. qPCR was conducted with the SYBR PrimeScript qPCR kit (Takara) in a CFX Connect™ qPCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. All samples were analyzed in triplicate. Fold changes of ALK or lincROR were calculated by the relative quantification (2^−ΔΔCt) method.

Total RNA was extracted from cultured cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions. The RNAs were reversely transcribed into complementary DNA (cDNA) using the RevertAidTM H Minus First Strand cDNA synthesis Kit (Takara, Otsu, Japan). As for miRNAs, the cDNA was synthesized using miScript Reverse Transcription Kit (Qiagen). qPCR was performed using the SYBR PrimeScript qPCR kit (Takara) in a CFX Connect™ qPCR Detection System (BIO-RAD Laboratories, Inc., Berkeley, CA, USA) according to the manufacturer’s instructions. U6 for miR-125a and GAPDH for STAT3 and HAS1 were separately used as internal control. The specific primers were as follow: miR-125a forward 5′-GCGACTCCCTGAGACCCTTTAA-3′ and universal primer 5′-GCGAGCACAGAATTAATACGAC-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and universal primer 5′-GCGAGCACAGAATTAATACGAC-3′; STAT3, forward 5′-GGAGGAGGCATTCGGAAAG-3′ and reverse, 5′-TCGTTGGTGTCACACAGAT-3′; HAS1, forward 5′-GTAGGGGCTGTTGGTGGGGAC-3′ and reverse 5′-TGAGCATGCGGTTGGTGAGGT-3′; GAPDH, forward 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. All reactions were performed in triplicate.