Gene Expression Quantification in Arabidopsis

Seedlings were frozen in liquid nitrogen and ground with glass beads using a Silamat S5 device (Ivoclar Vivadent) for 10 s. RNA was extracted using TRIzol reagent and treated with DNAse I according to manufacturer instructions (ThermoFisher Scientific). One µg of total RNA was used as template for reverse transcription using iScript Reverse Transcription Supermix as recommended by the manufacturer (BioRad). qPCR was performed with iTaq Universal SYBR®Green Supermix (BioRad) and primers listed in Supplementary Table 1, in a CFX384 machine (BioRad). Gene expression levels were compared to the reference gene GAPC-2 (At1g13440), ACT2 (At3g18780) or IPP2 (At3g02780).

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Vergara Z., Gomez M.S., Desvoyes B., Sequeira-Mendes J., Masoud K., Costas C., Noir S., Caro E., Mora-Gil V., Genschik P, & Gutierrez C. (2023). Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition. Nature Communications, 14, 1270.

Publication 2023

Silamat S5 DNAse I iScript Reverse Transcription Supermix iTaq Universal SYBR®Green Supermix CFX384 machine

Act2 Device Dnase i Frozen Gene Gene expression Ivoclar Nitrogen Primers Reverse transcription Seedlings Sybr green Trizol Vivadent

Corresponding Organization :

Other organizations : Institut de Biologie Moléculaire des Plantes

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Variable analysis

independent variables

Freezing of seedlings in liquid nitrogen
Grinding of seedlings with glass beads using a Silamat S5 device for 10 s

dependent variables

Gene expression levels

control variables

RNA extraction using TRIzol reagent
DNAse I treatment of RNA according to manufacturer instructions
Reverse transcription of 1 µg of total RNA using iScript Reverse Transcription Supermix
QPCR performed with iTaq Universal SYBR®Green Supermix and primers listed in Supplementary Table 1
Comparison of gene expression levels to reference genes GAPC-2 (At1g13440), ACT2 (At3g18780), or IPP2 (At3g02780)

positive controls

Not specified

negative controls

Not specified

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