Reverse transcription and qRT-PCR
Total RNA from plant tissues was isolated using the Total RNA Extraction
Kit (iNtRON Biotechnology, Korea). For reverse transcription (RT),
first-strand cDNAs were prepared with 5 μg of total RNA using M-MLV
reverse transcriptase and oligo 15 primer (Promega) and diluted to 50 μL
with miniQ water. Gene expression levels were measured by qPCR analysis.
Around 20 μL of qPCR mixture containing 2 μL of first-strand cDNAs, 10
μL of 2X QuantiTect SYBR Green I Master (Roche, Switzerland), and 0.25
mM of the forward and reverse primers for each gene was used. The qPCR
analysis was performed using the Light Cycler 2.0 (Roche Diagnostics,
Switzerland). The relative expression of each gene was normalized with
glyceraldehyde phosphate dehydrogenase (GADPH) as an internal control.
The gene-specific primers used for qPCR analysis are listed in Table
S3-4.