Abi stepone plus pcr instrument

A549 and H1299 cells were treated with different final concentrations of EGF (0, 2.5, 5, 10, 20 or 50 ng/ml) for 48 h. Total RNA was extracted from these A549 and H1299 cells using RNAiso Plus (Takara Bio, Otsu, Japan) according to the manufacturer's protocol and was quantified with NanoDrop 2000 (Thermo Fisher Scientific). RNA (1 µg) was used as the template for cDNA synthesis; cDNA was reverse transcribed with the Primscript RT Reagent kit (Takara Bio). RT-qPCR reactions were performed on ABI StepOnePlus PCR instrument (Applied Biosystems; Thermo Fisher Scientific) for 40 cycles at 95°C for 5 sec, and at 60°C for 30 sec. Comparative quantification was determined using the 2^−ΔΔCT method. Expression levels of RFPL3 and hTERT mRNA were standardized to GAPDH. Primer sequences are listed in Table I.

Total RNA was extracted from cerebral ischemic penumbra tissues using TRIzol reagent (Invitrogen, Frederick, MD, USA) according to the manufacturer’s instructions. Then, RNA was reverse-transcribed into cDNA using a PrimeScript Reagent Kit (Vazyme, Nanjing, China). Quantitative real-time PCR was implemented on an ABI StepOne Plus PCR instrument (Applied Biosystems, CA, USA) with an SYBR green kit (Applied Biosystems). The relative gene expression levels were quantified and normalized to GAPDH. The primer sequences used were as follows:
Nos2: F: CAGCTGGGCTGTACAAACCTT and R: CATTGGAAGTGAAGCGTTTCG.
Il6: F:GCTGGTGACAACCACGGCCT and R: AGCCTCCGACTTGTGAAGTGGT;
Tnfa: F: CAAGGGACAAGGCTGCCCCG and R: GCAGGGGCTCTTGACGGCAG;
Ccl2:F: TTGAGGTGGTTGTGGAAAAGG and R: GTGCTGACCCCAAGAAGGAAT;
Il1b:F: AAGCCTCGTGCTGTCGGACC and R: TGAGGCCCAAGGCCACAGG;
Il10: F: GGTTGCCAAGCCTTATCGGA and R: ACCTGCTCCACTGCCTTGCT;
Gapdh: F: GCCAAGGCTGTGGGCAAGGT and R: TCTCCAGGCGGCACGTCAGA.