We designed degenerate primer according to the MADS-box conserved region and isolated two cDNA from rice, FDRMADS6 and FDRMADS7, which are homologous to AP1.
Single strand cDNA was synthesized by using the 'TaqMan Reverse Transcription Reagents' (Applied Biosystems, Inc., Loughborough, UK) and oligo dT primer according to the manufacturer's instructions.
Total RNA were isolated from 100 mg of roots, stems, leaves, petals and treated leaves and reverse transcribed to the first-strand cDNAs with an oligo (dT) primer according to the manual of PrimeScript® RT reagent Kit (TaKaRa, Japan).
One microgram of total RNA was used for reverse transcription reaction with H Minus M-MuLV Reverse Transcriptase (Fermentas) and 3'-terminal primer, according to the manufacturer's protocol.
First strand cDNA synthesis was performed with Superscript III Reverse Transcriptase (Invitrogen, CA) and a polyT22 primer according to the manufacturer's instructions.
cDNA templates were synthesized from 1 µg of total RNA, using SuperScript II reverse transcriptase and an oligo dT) primer according to the manufacturer's instructions.
The first strand of cDNA was synthesized using Super Script™ II Reverse Transcriptase (Invitrogen) with oligo dT) primer, according to the manufacturer's instructions.
Reactions were set up in 25 µl, using 50 ng of cDNA and 70 nM of each primer, according to the manufacturer's protocol.
Total RNA was extracted from specimens with TRI-LS (Sigma-Aldrich), and was reverse-transcribed with the Transplex whole transcriptome amplification (WTA) kit (Sigma-Aldrich) [21] using a quasi-random primer, according to the manufacturer's protocol.
Real-time PCR was performed with an iCycler (Bio-Rad) using the iQSYBR Green PCR supermix (Bio-Rad) with miRNA specific primers and a universal qPCR primer according to the manufacturer's protocol for the NCode Kit (Table S1).
Single-stranded cDNAs were synthesized from total RNAs (5 µg) from various tissues using Superscript II reverse transcriptase (Gibco BRL, Invitrogen) with the oligo dT 18 primer according to the manufacturer's instructions.
