Fast sybr green master mix kit

Total RNA and small RNA were prepared from petals at the three following developmental stages: Non-colored petals early during development (Closed bud; Stage 1); Petals at onset of anthocyanin synthesis (Closed bud; Stage 2); Fully colored petals with maximum anthocyanin content (Bud opening; Stage 3). Total RNA was prepared as previously described69 (link). One microgram of RNA was used in reverse transcription assay and qPCR as previously described70 (link) using gene specific primers (Supplementary Notes 10; Supplementary Tables 8 and 9). Small RNAs were extracted using Macherey-Nagel NucleoSpin® miRNA. Contaminating DNA was removed using the Ambion® DNA-free kit (Cambridgeshire, UK). RNA concentration was measured on a NanoDrop ND-1000 Micro-Volume (NanoDrop Technologies) before and after DNase treatment. Small RNA quantification was performed using stem-loop RT-PCR as previously described71 . Reverse transcription was performed with RevertAid kit (Thermo Fisher Scientific). Primers specific to 5.8S rRNA or stem-loop RT-primer for miR156 (Supplementary Notes 10; Supplementary Table 8) were used. 5.8S rRNA and miR156 expression were quantified on QuantStudio™ 6 Flex Real-Time PCR 384 (Applied Biosystems) using Fast SYBR® Green Master Mix kit (Roche Diagnostic) and specific primers (Supplementary Notes 10). Data were collected for three independent biological replicates.