Protocol detail

Gene Expression Analysis via RT-qPCR

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Gene expression analysis was performed according to method of Jin et al. (2015) (link). Total RNA was extracted with an RNA extraction kit (TRIzol reagent; Invitrogen), then the first-strand cDNA was synthesized using 3 μg of RNA and 200U M-MLV reverse transcriptase (Invitrogen). Real-time PCR was performed using an optical 96-well plate in an ABI Stepone plus PCR system (Applied Biosystems) by using SYBR Premix reagent F-415 (Thermo Scientific). OsUBI was used as an endogenous control (Supplementary Table S1 at JXB online).

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Qu L., Wu C., Zhang F., Wu Y., Fang C., Jin C., Liu X, & Luo J. (2016). Rice putative methyltransferase gene OsTSD2 is required for root development involving pectin modification. Journal of Experimental Botany, 67(18), 5349-5362.

Publication 2016

TRIzol reagent M-MLV reverse transcriptase ABI Stepone plus PCR system SYBR Premix reagent F-415

Cdna Gene expression analysis Real time pcr Reverse transcriptase Trizol

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Variable analysis

independent variables

RNA extraction method (TRIzol reagent; Invitrogen)
First-strand cDNA synthesis (using 3 μg of RNA and 200U M-MLV reverse transcriptase; Invitrogen)
Real-time PCR method (using an optical 96-well plate in an ABI Stepone plus PCR system (Applied Biosystems) and SYBR Premix reagent F-415 (Thermo Scientific))

dependent variables

Gene expression levels

control variables

OsUBI as an endogenous control for real-time PCR

controls

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

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