RT-PCR Analysis of mRNA Expression

Total RNA was extracted from A549 cells using the TaKaRa FastPure RNA kit (Takara Bio, Ohtsu, Japan), according to the manufacturer’s instructions. First-strand synthesis and RT-PCR were performed using the One-step SYBR PrimeScript RT-PCR kit (TAKARA, Ohtsu, Japan), according to the manufacturer’s protocol. Amplification and detection were performed using the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems, Foster City, CA). PCR primers were purchased from Qiagen. The change in expression of each target mRNA was calculated relative to the level of 18S rRNA47 (link)48 (link).

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Daijo H., Hoshino Y., Kai S., Suzuki K., Nishi K., Matsuo Y., Harada H, & Hirota K. (2016). Cigarette smoke reversibly activates hypoxia-inducible factor 1 in a reactive oxygen species-dependent manner. Scientific Reports, 6, 34424.

Publication 2016

TaKaRa FastPure RNA kit One-step SYBR PrimeScript RT-PCR kit Applied Biosystems 7300 Real-time PCR System PCR primers

A549 cells Mrna Primers Rt pcr Synthesis

Corresponding Organization :

Other organizations : Kyoto University Hospital, Kansai Medical University, Kyoto University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Change in expression of each target mRNA

control variables

18S rRNA

controls

Positive control: Not mentioned
Negative control: Not mentioned

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