Cfx manager software system

Quantification of RNA was performed with a nanodrop spectrophotometer (Nanodrop Technologies) or with a Qubit 2.0 fluorometer (Invitrogen) using the Qubit RNA BR assay kit (Molecular Probes). cDNA was reverse transcribed from RNA with primers listed in Supplementary Table S1. Real-time PCRs were followed with a Bio-Rad CFX Connect Real-Time PCR detection system using iTaq Universal SYBR Green Supermix (Bio-Rad). Reactions were initiated by incubating the samples at 95°C for 3 min to activate Taq polymerase, followed by 40 cycles of 10 s at 95°C and 30 s at 55°C. Melting-curve analysis was performed starting at 65°C with stepwise temperature elevations of 0.5°C every 5 s to check for non-specific products. PCR primer sequences used to amplify the different genes assayed are listed in Supplementary Table S1. Reactions contained 7.5 μl of 2× SYBR iTaq Universal SYBR Green Supermix (Bio-Rad), 5 μl of cDNA (0.2 ng μl − 1) and 250 nm of each product-specific primer in a final volume of 15 μl. Data were analyzed using the CFX Manager Software system (Bio-Rad). The results of the quantitative RT-PCR analysis were normalized using three genes, At2g28390, At4g34270 and At5g25760, which have been shown to be superior reference genes for transcript normalization in Arabidopsis (29 (link)).

The same RNA used for RNA-seq was used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. cDNAs were synthesized from 1µg of RNA using the SuperScript IV Reverse Transcriptase (Invitrogen) kit and random hexamers following the provided protocol. Real-time PCR was performed in the Bio-Rad CFX Connect Real-Time System with Syber Green master mix (Bio-Rad, Hercules, CA, USA) following the provided protocol. Each sample was run in triplicate. Three biological samples were run for N2 (wild-type), daf-1(m40), and daf-1(m40); daf-3(mgDf90) double mutants. The PCR protocol used was: 95 °C for 2 min; 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers used are listed in Table S2. The relative expression fold change was calculated in the CFX manager software system (Bio-Rad) using the 2^^−ddCT method. cdc-42 was used as a control.