Quantifying RNA Dynamics During Flower Development

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.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Raymond O., Gouzy J., Just J., Badouin H., Verdenaud M., Lemainque A., Vergne P., Moja S., Choisne N., Pont C., Carrère S., Caissard J.C., Couloux A., Cottret L., Aury J.M., Szecsi J., Latrasse D., Madoui M.A., François L., Fu X., Yang S.H., Dubois A., Piola F., Larrieu A., Perez M., Labadie K., Perrier L., Govetto B., Labrousse Y., Villand P., Bardoux C., Boltz V., Lopez-Roques C., Heitzler P., Vernoux T., Vandenbussche M., Quesneville H., Boualem A., Bendahmane A., Liu C., Le Bris M., Salse J., Baudino S., Benhamed M., Wincker P, & Bendahmane M. (2018). The Rosa genome provides new insights into the domestication of modern roses. Nature genetics, 50(6), 772-777.

Publication 2018

NanoDrop ND-1000 Micro-Volume RevertAid kit QuantStudio™ 6 Flex Real-Time PCR 384 Fast SYBR® Green Master Mix kit

5 8s rrna Anthocyanin Assay Biological Diagnostic Dnase Fast green Gene Mirna Primers Real time pcr Reverse transcription Rrna Rt pcr Stem Synthesis

Corresponding Organization :

Other organizations : Université Claude Bernard Lyon 1, École Normale Supérieure de Lyon, Centre National de la Recherche Scientifique, Laboratoire de Reproduction et Développement des Plantes, Université de Toulouse, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Genoscope, Université Paris-Saclay, Génétique, Diversité, Écophysiologie des Céréales, Institut des Sciences des Plantes de Paris Saclay, Sorbonne Paris Cité, Université Paris Cité, Huazhong Agricultural University, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, École Nationale des Travaux Publics de l'État, Aix-Marseille Université, Institut de Recherche pour le Développement, Institut de Biologie Moléculaire des Plantes, University of Tübingen, Université d'Évry Val-d'Essonne

Top 5 similar protocols

Variable analysis

independent variables

Developmental stage of petals: 1) Non-colored petals early during development (Closed bud; Stage 1), 2) Petals at onset of anthocyanin synthesis (Closed bud; Stage 2), 3) Fully colored petals with maximum anthocyanin content (Bud opening; Stage 3)

dependent variables

Total RNA expression
Small RNA expression
Expression of 5.8S rRNA
Expression of miR156

control variables

RNA concentration was measured on a NanoDrop ND-1000 Micro-Volume before and after DNase treatment to ensure removal of contaminating DNA

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!