Rotor gene q 2plex real time pcr machine

For qRT-PCR analysis, leaf samples were collected from the full growth of 4^th leaf stage. Total RNA was extracted from 100 mg of leaf tissue using the RNeasy Plant Mini Kit (Qiagen, CA, USA). Single-stranded cDNA was synthesized from 500 ng of total RNA using TOP-script Reverse Transcriptase (Enzynomics, Korea). eEF-1α was used as a housekeeping gene to normalize the three E. crus-galli accessions. Primers for 19 genes (2 Serine/threonine receptor kinases, 4 Leucine-rich repeat receptor kinases, 5 Cysteine-rich protein kinases, and 8 Calcineurin B-like kinases) were used (S1 Table). qRT-PCR reactions were carried out using TOP-realTM qPCR 2X PreMIX for SYBR Green (Enzynomics, Korea) with a final volume of 20 μl in a Rotor-Gene Q 2plex real-time PCR machine (Qiagen, CA, US). The qRT-PCR cycle program was as follows: (1) 95°C for 15 min for initial denaturation, (2) 95°C for 10 sec for denaturation, (3) 55°C for 15 sec for annealing, (4) 72°C for 20 sec for elongation, and then the cycles from (2)-(4) were repeated 45 times. Relative quantification of each single gene expression was evaluated using the delta-delta Ct method [49 (link)].