The method was as described previously 27 (link),28 (link). Briefly, A549 and NCI-H460 cells were respectively treated with CyH (0, 1.51, 3.13, 6.25, 12.5, and 25 μM) for 16 h. Total RNA in the treated and untreated cells was extracted using Trizol reagent and the mRNA levels of the target genes were analyzed using One Step SYBR® PrimeScript® RT-PCR (TaKaRa, China). All primers were synthesized by Sangon Biotech (Shanghai, China) and the sequences were listed in Table 1 . The reaction conditions were as follows: 42 °C for 5 min, then 95 °C for 10 s, followed by 40 cycles (95 °C for 5 s and 60 °C for 31 s). β-actin was used as a control to normalize the relative mRNA levels of all target genes.
One step sybr primescript rt pcr
Manufactured by Takara Bio
Sourced in China
One Step SYBR PrimeScript RT-PCR is a real-time PCR kit that combines reverse transcription and PCR amplification in a single step. It utilizes SYBR Green I dye for detection of amplified DNA products.
Automatically generated - may contain errors
5 protocols using one step sybr primescript rt pcr
1
RNA Expression Analysis of A549 and NCI-H460 Cells
2
Quantitative analysis of HIF-1α, VEGF, and IL-8 mRNA
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The method was as described previously [26] (link). Briefly, total RNA was extracted by homogenization in 1 mL TRIZOL Reagent (Invitrogen), followed by chloroform extraction and isopropanol precipitation. The analysis of HIF-1α, VEGF, and IL-8 mRNA relative levels was performed using One Step SYBR PrimeScript RT-PCR (TaKaRa, China) according to the manufacturer’s instructions. A 50 ng sample of total RNA from A549 or NCI-H460 cells was used. The primers used were as follows: for human HIF-1α: forward 5′-TCTGGGTTGAAACTCAAGCAACTG-3′ and reverse 5′-CAACCGGTTTAAGGACACATTCTG-3′ [Genbank: NM_001243084.1]; VEGF: forward5′-TCTACCTCCACCATGCCAAGT-3′ and reverse 5′-GATGATTCTGCCCTCCTCCTT-3′ [Genbank: NM_001025366.2]; IL-8: forward 5′-TTGCCAAGGAGTGCTAAAGAA-3′ and reverse 5′-GCCCTC TTCAAAAACTTCTCC-3′ [Genbank: NM_000584.3]; β-actin: forward 5′-TCAAGATCATTGCTCCTCCTG-3′ and reverse 5′-CTGCTTGCTGATCCACATCTG-3′ [Genbank: NM_001017992.3]. All the primers were synthesized by TaKaRa Biotechnology (Dalian,) Co., LTD (Dalian, China). The thermocycling conditions were as follows: 42°C for 5 min, 95°C for 10 s, followed by 40 cycles at 95°C for 5 s, and 60°C for 31s. The relative HIF-1α, VEGF, and IL-8 mRNA levels were normalized to β-actin. The experiment was repeated in triplicate.
3
Quantitative Analysis of HIF-1α and VEGF Regulation
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The method was as described previously 28 (link)-30 (link). Briefly, A549 and H460 cells were treated with CyH (0, 6.25, 12.5, and 25 μM) for 24 h. Total RNA from the cells was extracted with TRIZOL® Reagent (Invitrogen; Thermo Fisher Scientifc, Inc.) according to the manufacturer's protocol. RT-qPCR analysis of HIF-1α, VEGF, and β-actin mRNA levels was performed using One Step SYBR® PrimeScript® RT-PCR (TaKaRa, China). The specific primers synthesized by Sangon Biotech (Shanghai, China) were as follows: HIF-1α forwards 5′-TCTGGGTTGAAACTCAAGCAACTG-3′ and reverse 5′-CAACCGGTTTAAGGACACATTCTG-3′ [Genbank: NM_001243084.1]; VEGF forwards 5′-TGCTTCTGAGTTGCCCAGGA-3′ and reverse 5′-TGGTTTCAATGGTGTGAGGACATAG-3′ [Genbank:NM_001287044.1]; β-actin forwards 5′-TCATGAAGTGTGACGTGGACATCCGT-3′ and reverse 5′-CCTAGAAGCATTTGCGGTGGACGATG-3′ [Genbank:NM_001101.4]. The cycling conditions were as follows: 42 °C for 5 min, 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s, and 60 °C for 31 s. Target gene mRNA levels were normalized to β-actin by using 2-∆∆Ct.
4
Quantitative RT-PCR Analysis of VEGF and IL-8 mRNA
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The mRNA levels of VEGF and IL-8 were detected by quantitative real-time PCR using One Step SYBR PrimeScript RT-PCR (TaKaRa, China) according to the manufacturer’s instructions. The method was as described previously (7 (link)). Total RNA was extracted from treated and untreated NSCLC cells with TRIzol reagent (Invitrogen). The following specific primers were used: human VEGF (forward 5′-TCTACCTCCACCATGCCAAGT-3′ and reverse 5′-GATGATTCTGCCCTCCTCCTT-3′) (Genbank: NM_001025366.2); IL-8 (forward 5′-TTGCCAAGGAGTGCTAAAGAA-3′ and reverse 5′-GCCCTCTTCAAAAACTTCTCC-3′) (Genbank: NM_000584.3); β-actin (forward 5′-TCAAGATCATTGCTCCTCCTG-3′ and reverse 5′-CTGCTTGCTGATCCACATCTG-3′) (Genbank: NM_001017992.3). All the primers were produced by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The thermocycling conditions for real-time PCR were as follows: 42°C for 5 min, 95°C for 10 s, followed by the PCR process (40 cycles at 95°C for 5 s, and 60°C for 31 s). The relative mRNA levels were normalized to β-actin. The experiment was repeated in triplicate.
5
Quantitative Analysis of CD36 Expression
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Each RNA of HAEC or THP-1 cell was manually extracted using TRIzol (Invitrogen, Waltham, MA, USA). cDNA and real-time PCR were performed with the One Step SYBR PrimeScript RT-PCR (TaKaRa, Japan) kit according to the manufacturer's instructions. Primers for human CD36 and β-actin as an internal control are as follows: human CD36 forward, 5′-TCTTTCCTGCAGCCCAATG-3′, reverse, 5′-AGCCTCTGTTCCAACTGATAGTGA-3′; human β-actin forward, 5′-GGGRCAGAAGGATTCCTATG-3′, reverse, 5′-GGTCTCAAACATGATCTGGG-3′. ABI StepOne Plus (Applied Biosystems, USA) was used for this quantitative real-time PCR reaction. The step for reverse transcription was done at 42°C for 5 min and at 95°C for 10 sec. The PCR amplification step was performed at 40 cycles of denaturation at 95°C for 5 sec and annealing and extension at 60°C for 60 sec. Each sample was performed in triplicate with no template controls and analyzed.
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