RNA Extraction and RT-qPCR for Gene Expression

Around 10⁹ cells were harvested for each sample and washed once with pre-chilled diethyl pyrocarbonate (DEPC)-treated water. Cell pellets were snap-frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Total RNA was obtained using the Trizol reagent (Invitrogen). RNA was then reverse transcribed using the PrimeScript RT kit (Takara, RR014A). The cDNA library was used as a template in real-time PCR using the Takara SYBR Premix Ex-Taq (Tli RNase H Plus) kit (Takara, RR420A). The primers for CLN2 and ACT1 were the same as in our previous study (Zhang Z. et al., 2017 (link)).

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Li P., Liu X., Hao Z., Jia Y., Zhao X., Xie D., Dong J, & Zeng F. (2020). Dual Repressive Function by Cip1, a Budding Yeast Analog of p21, in Cell-Cycle START Regulation. Frontiers in Microbiology, 11, 1623.

Publication 2020

Trizol reagent PrimeScript RT kit SYBR Premix Ex-Taq (Tli RNase H Plus) kit

Cdna library Cell Diethyl pyrocarbonate Frozen Nitrogen Pellets Primers Real time pcr Rnase h Trizol

Corresponding Organization :

Other organizations : Hebei Agricultural University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels of CLN2 and ACT1 genes

control variables

Cell number (~10^9 cells) for each sample
Washing cells with pre-chilled DEPC-treated water
Snap-freezing cell pellets in liquid nitrogen and storing at -80°C before RNA extraction
RNA extraction using Trizol reagent
Reverse transcription using PrimeScript RT kit
Real-time PCR using Takara SYBR Premix Ex-Taq (Tli RNase H Plus) kit
Primer sequences for CLN2 and ACT1 genes were the same as in a previous study (Zhang Z. et al., 2017)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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