|
| Status |
Public on May 27, 2013 |
| Title |
apices_2 weeks + 4 weeks Vernalization_Repl 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
main shoot apex enriched
|
| Organism |
Arabis alpina |
| Characteristics |
accession: Pajares
vernalization treatment: 4°C, short day (8 hours light/16 hours dark)
zt at harvest: 8
developmental stage: reproductively incompetent (2 weeks in long days) + non inductive vernalization. Vegetative
|
| Treatment protocol |
Vernalization treatments were performed at 4°C and in SD conditions (8 h light/16 h dark).
|
| Growth protocol |
Arabis alpina Pajares plants were grown in control conditions in LD (16 h light/8 h dark). Light was provided by fluorescent tubes complemented by incandescent bulbs to increase the proportion of far red light. The temperatures were ranging from 20°C during the day to 18°C during the night unless differently indicated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extract using RNeasyTM (Qiagen), double column purification performed. Genomic DNA was afterwards digested using the DNA-freeTM Kit (Ambion). RNA quality and integrity were measured with the Bioanalyzer (Agilent).
|
| Label |
Cy5
|
| Label protocol |
The Agilent Quick Amp Labeling kit was used to synthesise Cy3 and Cy5 labeled cRNA. For each RNA sample, 500 ng were used for the labelling. Afterwards the concentration and incorporation of the cRNA and dyes were measured with the Nanodrop. For the hybridization, 2000 ng of Cy3 and Cy5 labeled cRNA were used for further fragmentation and hybridization to the custom microarray (Labelling and hybridizations were performed at Service XS, Leiden, NL).
|
| |
|
| Channel 2 |
| Source name |
main shoot apex enriched
|
| Organism |
Arabis alpina |
| Characteristics |
accession: Pajares
reference: equal amounts of RNA from each individual sample
|
| Treatment protocol |
Vernalization treatments were performed at 4°C and in SD conditions (8 h light/16 h dark).
|
| Growth protocol |
Arabis alpina Pajares plants were grown in control conditions in LD (16 h light/8 h dark). Light was provided by fluorescent tubes complemented by incandescent bulbs to increase the proportion of far red light. The temperatures were ranging from 20°C during the day to 18°C during the night unless differently indicated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extract using RNeasyTM (Qiagen), double column purification performed. Genomic DNA was afterwards digested using the DNA-freeTM Kit (Ambion). RNA quality and integrity were measured with the Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
The Agilent Quick Amp Labeling kit was used to synthesise Cy3 and Cy5 labeled cRNA. For each RNA sample, 500 ng were used for the labelling. Afterwards the concentration and incorporation of the cRNA and dyes were measured with the Nanodrop. For the hybridization, 2000 ng of Cy3 and Cy5 labeled cRNA were used for further fragmentation and hybridization to the custom microarray (Labelling and hybridizations were performed at Service XS, Leiden, NL).
|
| |
|
| |
| Hybridization protocol |
Temperature of hybridization : 60°C. Hybridization times, washing condition and scanning of the arrays were performed according to the Agilent protocols.
|
| Scan protocol |
Images were quantified using Agilent Feature Extraction Software (version 9.5).
|
| Description |
Biological replicate 4 of 4, every replicate was a pool of apices from plants growed under the same conditions. Pooled plants were omogenized in liquid nitrogen before RNA extraction
mix of all the above 16 biological replicates, after total RNA extraction, equal amounts of RNA from each individual sample were pulled. On the same array one biological replicate and the reference pool were hybridized.
|
| Data processing |
To obtain a representative gene expression value for each gene in each array we corrected for the back ground signal, normalized the intensity distribution, filtered low intensity probes and summarized the probes into one single value. To normalize the data we first normalized the log-transformed intensity distribution within each array using intensity dependent normalization (function “normalizeWithinArrays”, method “loess”, R package “limma”). To make the values comparable between arrays we applied a quantile normalization taking advantage of the common reference channel (function “normalizeBetweenArrays”, method “Gquantile”, R package “limma”). We then excluded probes with maximum intensity lower than 7.3 in the common reference channel of all 16 arrays. This threshold was based on the 75th quantile of the intensity distribution observed for intron coding probes which served as a control for the interspecific hybridization. In total 27607 (91%) genes have at least one 60-mer exon coding probe with a maximum expression above 7.3 in the common reference channel. The summarization step for genes with multiple probes was performed using the Robust Multichip Average (RMA) on the normalized log-transformed red channel intensities. In brief, The RMA procedure uses the median-polishing algorithm to find a robust median expression value for each gene on each array.
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| |
|
| Submission date |
Aug 29, 2012 |
| Last update date |
May 29, 2013 |
| Contact name |
Emiel Ver Loren van Themaat |
| E-mail(s) |
themaat@mpiz-koeln.mpg.de
|
| Organization name |
Max Planck Institute
|
| Street address |
Carl von linneweg
|
| City |
Cologne |
| ZIP/Postal code |
50823 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16007 |
| Series (1) |
| GSE40455 |
Mechanisms of age-dependent response to winter temperature in perennial flowering of Arabis alpina. |
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