Nucleospin rna plant mini spin kit

The expression rates of Arabidopsis genes (NIA1, NIA2, GLB1, GLB2, GSNOR1, CCD7, CCD8, D14, MAX1, MAX2) were determined by quantitative real-time reverse transcription PCR (RT-qPCR). RNA was purified from 90 mg of 7-day-old seedlings by using a NucleoSpin RNA Plant mini spin kit (Macherey-Nagel) according to the manufacturer's instruction. Furthermore, an additional DNAase digestion and purifying step was applied (ZYMO Research), and cDNA was synthetized using RevertAid reverse transcriptase. Primer3 software was used for designing primers. The primers used for RT-qPCR analyses are listed in Table S1. The expression rates of the NO- and SL associated genes were detected by quantitative real time PCR machine (qTOWER 2.0, Jena Instruments) using SYBR Green PCR Master Mix (Thermo Mix) (Gallé et al., 2009 (link)). Data were analyzed by using qPCRsoft3.2 software (Jena Instruments). Data were normalized to the transcript levels of the control samples; ACTIN2 (At3918780) and GAPDH2 (At1913440) were used as internal controls (Papdi et al., 2008 (link)). Each reaction was carried out in three replicates using cDNA synthesized from independently extracted RNAs. These analyses were performed on three separate plant generations with three technical replicates in each (n = 3).

The expression rate of AtROP2 gene was determined by quantitative real-time reverse transcription-PCR (RT-qPCR). RNA was purified from 90 mg root tissue by using a NucleoSpin RNA Plant mini spin kit (Macherey–Nagel) according to the manufacturer’s instruction. An additional DNase digestion was applied (by using a DNA Clean and Concentrator Kit from Zymo Research and DNase I from Thermo Scientific), and cDNA was synthetized using RevertAid reverse transcriptase (Thermo Scientific). Primers were designed for the selected coding sequences using the Primer3 software; the primers used are listed in Table S1. The expression rate was monitored using the SYBR Green PCR Master Mix (Thermo Scientific) as described by Gallé et al. [64 (link)]. Data analysis was performed using qPCRsoft3.2 software (Analytik Jena AG, Jena, Germany). ACTIN2 (At3g18780) and GAPDH2 (At1g13440) genes were used as internal controls.