Quantitative Real-Time RT-PCR for Rice Gene Expression

Samples were collected and immediately frozen in liquid nitrogen, then stored at –80°C. Total RNA was extracted using TRIzol reagent (Invitrogen). After RNase-free DNase treatment, 5 μg of RNA was used for cDNA synthesis using M-MLV reverse transcriptase (Promega) in a 50-μl reaction mixture. The quantitative, real-time reverse transcription polymerase chain reaction (qRT-PCR) technique was performed using 2×SYBR Green Master Mix reagent (Bio-Rad) in a 96-well plate using a Bio-Rad CFX96 real-time PCR system. Three technological replicates were used for each biological sample. Six rice reference genes (from our unpublished data on the selection of stable reference genes to normalise gene expression in rice) were assessed for their potential as stable internal standards by geNorm as previously described (Vandesompele et al., 2002 (link)). These genes were TI (LOC_Os01g05490), ARF (LOC_Os05g41060), EF-1α (LOC_Os03g08020), UBC (LOC_Os02g42314), Profilin-2 (LOC_Os06g05880), and Actin1 (LOC_Os03g50885). As a result of this analysis, UBC, Profilin-2, and Actin1 were selected as internal standards for all leaf samples (Supplementary Figure S1). All primers used for qRT-PCR analysis are listed in Supplementary Table S3, with good PCR efficiencies (85–105%) assessed using a 10-fold dilution series of total cDNA.