RNA Extraction and RT-PCR Analysis

Total RNA was extracted from cells using the RNeasy™ Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions^{6 (link)}. First-strand synthesis and RT-PCR were performed using the QuantiTect™ Reverse Transcription Kit (Qiagen) and Rotor-Gene™ SYBR Green PCR Kit (Qiagen), according to the manufacturer’s protocol. Amplification and detection were performed using Rotor-Gene™ Q (Qiagen). PCR primers were purchased from Qiagen. The change in expression of each target mRNA was calculated relative to the level of 18S rRNA.

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Sumi C., Okamoto A., Tanaka H., Kusunoki M., Shoji T., Uba T., Adachi T., Iwai T., Nishi K., Harada H., Bono H., Matsuo Y, & Hirota K. (2018). Suppression of mitochondrial oxygen metabolism mediated by the transcription factor HIF-1 alleviates propofol-induced cell toxicity. Scientific Reports, 8, 8987.

Publication 2018

RNeasy™ Mini Kit QuantiTect™ Reverse Transcription Kit Rotor-Gene™ SYBR Green PCR Kit Rotor-Gene™ Q PCR primers

18s rrna Gene Mrna Primers Reverse transcription Rt pcr Sybr green Synthesis

Corresponding Organization :

Other organizations : Kansai Medical University, Kyoto University, Research Organization of Information and Systems

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Protocol cited in 3 other protocols

Variable analysis

independent variables

RNA extraction method: RNeasy™ Mini Kit (Qiagen)
Reverse transcription method: QuantiTect™ Reverse Transcription Kit (Qiagen)
RT-PCR method: Rotor-Gene™ SYBR Green PCR Kit (Qiagen)

dependent variables

Change in expression of each target mRNA relative to the level of 18S rRNA

control variables

Manufacturer's protocols for RNA extraction, reverse transcription, and RT-PCR

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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