One step sybr primescript pcr kit

For quantitative reverse transcription PCR (qRT‒PCR) assays, total RNA was extracted from HNSCC cells using the Axygen Total RNA Isolation Kit and further reverse transcribed using the PrimeScript ™ RT Reagent Kit with gDNA Eraser (Takara, Japan). Then, synthesized cDNA was subjected to real-time PCR in the presence of a One Step SYBR^@ PrimeScript^@ PCR Kit (Takara, Japan) in an Applied Biosystems StepOne™ machine (Invitrogen, USA). The differential expression of target genes was evaluated and quantified using the formula 2^−ΔΔCT, with target gene expression levels normalized to that of the housekeeping gene GAPDH. The designed primer sequences used in the current research are listed in Supplementary Table 3.

Total RNAs were extracted from the cultured cells using Trizol reagent (TaKaRa, Japan) according to the manufacturer's protocol. Following template cDNAs were obtained by reverse transcription (RT) using PrimeScript reverse transcription kit (TaKaRa, Japan), real time PCR was performed empolying Thermal Cycler Dice Real Time System (Thermo, USA) utilizing a one-step SYBR PrimeScript PCR kit (TaKaRa, Japan). The transcripts in cDNAs samples were detected by PCR reactions with primer as follows: Ps1 forward, 5-′GCCAGAATGACAGCCAAGAAC-3′, reverse, 5-′GCTCTTCGTCTTCCTCCTCATC-3′; Bace1 forward, 5-′ GACCTCCGAAAGGGTGTGTAT-3′, reverse, 5-′ AGTCAAAGAAGGGCTCCAAAG-3′; GAPDH forward, 5-′GGTGAAGGTCGGTGTGAACG-3′, reverse, 5-′CTCGCTCCTGGAAGATGGTG-3′ (Sangon Biotech, China). The quantity in each sample was normalized to GAPDH.