Real-time quantitative polymerase chain reaction (qPCR)

BW Bour-Jr Wang
YC Yu-Ying Chen
HC Hui-Hsuan Chang
RC Rong-Jane Chen
YW Ying-Jan Wang
YL Yu-Hsuan Lee
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THP-1 cells were exposed to 10 or 12.5 μg/ml ZnO NPs. After 18 h exposure, the RNA was isolated using TOOLSharp RNA Extractor (TTD-NRNA200, BIOTOOLS Co., Ltd., Taipei, Taiwan). RNA concentrations and 260/280 nm ratios were measured by NanoDrop 2000 (Thermo Scientific™, Waltham, MA, USA). Reverse transcription (RT) was performed using TOOLSQuant II Fast RT Kit (KRT-BA06-2, BIOTOOLS Co., Ltd., Taipei, Taiwan). Finally, a mixture of the synthesized cDNA, primers and TOOLS 2 × SYBR qPCR Mix (FPT-BB05, BIOTOOLS Co., Ltd., Taipei, Taiwan) was carried out in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster, CA, USA) using SYBR Green Master Mix (Thermo Fisher Scientific). The average of the ΔΔC(t) of triplicate samples was calculated using β-actin as a housekeeping gene. The sequences of forward primers and reverse primers were summarized as described under Supplementary information.

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