The RNA was extracted from the yeast cells of the exponential growth period by the phenol-chloroform extraction method (50 (link)). The extracted RNA was treated with RNase-free deoxyribonuclease I (Takara, 2270A) to remove DNA and quantified by Nanodrop 2000 (Thermo Fisher Scientific). The RNA quality was determined by agarose gel electrophoresis. RNA (0.5 μg) was taken for complementary DNA (cDNA) synthesis in the NovoScriptPlus All-in-one First Strand cDNA Synthesis SuperMix (Novoprotein). The qPCR was carried out using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121). Primers used for RT-qPCR were described in table S2. 2−ΔΔCt was used to calculate the quantity of relative transcription level.
Protocol detail
Yeast RNA Extraction and qPCR Analysis
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Nanodrop 2000
NovoScriptPlus All-in-one First Strand cDNA Synthesis SuperMix
iTaq Universal SYBR Green Supermix
Agarose gel electrophoresis
Cdna
Chloroform
Deoxyribonuclease i
Phenol
Primers
Rnase
Sybr green
Synthesis
Transcription
Yeast
Corresponding Organization : Hubei University
Other organizations : Institute of Hydrobiology, Chinese Academy of Sciences
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Variable analysis
independent variables
- Phenol-chloroform extraction method
- Relative transcription level
- Exponential growth period of yeast cells
- RNase-free deoxyribonuclease I treatment to remove DNA
- RNA quantification by Nanodrop 2000
- RNA quality determination by agarose gel electrophoresis
- CDNA synthesis using NovoScriptPlus All-in-one First Strand cDNA Synthesis SuperMix
- QPCR using iTaq Universal SYBR Green Supermix
- Primers used for RT-qPCR as described in table S2
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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