Long Days: 16 h L / 8 hs D, 80 uE, 22 °C; Short Days: 8 h L / 16 h D, 160 uE, 22 °C
Extracted molecule
total RNA
Extraction protocol
Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
Label
Cy5
Label protocol
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
Hybridization protocol
Hybridization with indirectly labeled mRNA (SGED_SOP_6.1.1, SGED_SOP_6.2.1-4)
Scan protocol
Axon 4000B scanner. Both the 635nm (red, Cy5) and 532nm (green, Cy3) channels are scanned simultaneously at 100% laser power, the PMT set between 600 and 950. Slides are scanned at a resolution of 10micron
Description
Plants of Solanum tuberosum subsp. andigena, which tuberize only under SD conditions, were grown in growth chambers under non-inductive LD conditions and half of the plants were transferred to inductive SD conditions for 1 or 15 days, whilst the rest of the plants were kept as controls in LD. Transgenic potato plants with reduced phyB levels, which tuberize irrespective of photoperiod, were also grown in LD conditions. All the plants, irrespective of treatment, were harvested 14 hours after the beggining of the photoperiod (i.e. immediately before dusk for plants on LD, and 8 hours after dusk for the plants on SD). A similar experimental protocol was used with plants of N. tabacum cv Hicks that flower at the same time irrespective of photoperiod and the isogenic line N. tabacum cv Hicks carrying the Maryland Mammoth mutant allele, which flower only on SD (i.e. the plants were grown under LD for 55 or 41 days in LD and then transferred for 1 or 15 days to SD conditions, respectively, whilst control plants were grown 56 days in LD). Finally, Nicotiana silvestris plants that flower only on LD were grown in non-inductive SD and then half of the plants were transferred to inductive LD conditions for 1 or 15 days. In all cases we harvested only the leaves, the organ in which daylength perception takes place and photoperiodic responses are initiated. SD conditions in this experiments were 8 hours of light/ 16 hours of darkness, 160 uEinstein, 22 °C. LD conditions were 16 hours of light/ 8 hours of darkness, 80 uEinstein, 22 °C. RNA was extracted with TRIZOL.
Data processing
The TIFF images were quantified using Genepix 5.0. Local background was subtracted from the signal value and the data was normalized using the quantile method in the limma package of bioconductor.
Background (CH2_BACKGROUND) subtracted raw intensity (F532 Mean - B532 Media)
CH2_BACKGROUND
CH2 background median intensity (B532 Media)
FLAG
B: no flag, good spot; X: undetectable spot; M: flagged for diameter < 70 microns, the percentage of saturated pixels > 30% or not validated PCR product
