RNA Isolation and RT-qPCR Protocol

RNA isolation and real-time PCR were performed as in Reference [20 (link)]. The Spectrum Plant Total Kit (Sigma-Aldrich, St Louis, MO, USA) and DNaseI (Sigma-Aldrich, St Louis, MO, USA) were used for total RNA extraction. First-strand cDNA was synthetized, using the RevertAid M-MuLV Reverse Transcriptase Kit (Thermo Fisher Scientific, Vilnius, Lithuania), 1000 ng of RNA, and oligo(dT)18 primers. Real-time PCR was performed, using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, St Louis, USA ) and a Rotor-Gene 6000 thermal cycler (Corbett Research, Sydney, Australia), according to Reference [39 (link)], with reactions run in technical triplicates. Primer sequences and annealing temperatures were based on Reference [22 (link)] and are listed in Supplementary Table S1. The average of Ct values for WT and phot1phot2 (darkened and non-darkened leaves) were used as a calibrator for calculating relative gene expression levels. Normalization of gene expression levels was performed, using factors based on reference gene levels (UBC, UBQ19, and SAND), calculated with geNorm v3.4 [51 (link)].

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Eckstein A., Grzyb J., Hermanowicz P., Zgłobicki P., Łabuz J., Strzałka W., Dziga D, & Banaś A.K. (2021). Arabidopsis Phototropins Participate in the Regulation of Dark-Induced Leaf Senescence. International Journal of Molecular Sciences, 22(4), 1836.

Publication 2021

DNaseI RevertAid M-MuLV Reverse Transcriptase Kit SYBR Green JumpStart Taq ReadyMix Rotor-Gene 6000

Cdna Gene Gene expression Isolation M mulv Oligo Plant Primer Real time pcr Reverse transcriptase Sybr green

Corresponding Organization : Jagiellonian University

Other organizations : University of Gdańsk, University of Wrocław

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Variable analysis

independent variables

Darkening of leaves

dependent variables

Relative gene expression levels

control variables

RNA isolation and real-time PCR protocols from Reference [20]
Spectrum Plant Total Kit (Sigma-Aldrich, St Louis, MO, USA) and DNaseI (Sigma-Aldrich, St Louis, MO, USA) for total RNA extraction
RevertAid M-MuLV Reverse Transcriptase Kit (Thermo Fisher Scientific, Vilnius, Lithuania) for first-strand cDNA synthesis
SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, St Louis, USA) for real-time PCR
Rotor-Gene 6000 thermal cycler (Corbett Research, Sydney, Australia) for real-time PCR
Primer sequences and annealing temperatures based on Reference [22]
Reference genes used for normalization of gene expression levels: UBC, UBQ19, and SAND

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