|Public on Jun 27, 2008
|Upper pulvinus 2 min polysomal RNA biorep 2
|upper pulvinus_2min_Polysomal RNA_biorep 2
|Polysomal RNA extracted from upper pulvinus
Gravity stimulation for 2min
|Plants were gravity stimulated by turning the pots horizontally (90º reorientation). Harvesting of the most gravity competent pulvinus (second pulvinus above soil level) was done at the following time points: 2 min, 5 min, 15 min, 30 min and 60 min. The upper (slow elongation) and lower (fast elongation) halves of six pulvini were harvested per time point. Control plants were kept vertical (no gravity stimulation). Pulvini from the control plants were separated into left and right halves (simulating upper and lower harvesting of pulvini in the gravity stimulated plants).
|Maize (Zea mays strain B73) plants were cultivated in soil using 20cm diameter pots (two plants per pot, 6 plants per time point). Greenhouse conditions were as follow: natural light, 30ºC day and 26ºC night temperatures. Plants were grown to an age of ~6 weeks.
|Pulvini tissue was immediately frozen in liquid nitrogen after harvesting in the greenhouse. The harvested pulvini samples were then transferred to the laboratory and grinded to a fine powder in mortars and pestles using liquid nitrogen. This frozen powder was separated into aliquots, one used for extraction of total RNA, the other for isolation of polysomes and subsequent extraction of polysomal RNA. Polyribosomes were harvested as described in Davies, E. and S. Abe (1995). (Methods for isolation and analysis of polyribosomes. Methods Cell Biol 50: 209-22). Total RNA and polysome associated RNA was extracted using a RNeasy Plant Mini Kit (Qiagen).
|Complimentary RNA (cRNA) was produced according to Affymetrix Eukaryotic one-cycle target labeling assay as specified in the GeneChip Expression Analysis Technical Manual.
|Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Maize Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. Hybridization reactions to Affymetrix Maize GeneChip expression arrays were done using the services of Expression Analysis Inc.
|Scanning of the Affymetrix Maize GeneChip expression arrays were done using the services of Expression Analysis Inc.
|no additional info
|Data extraction was also performed by Expression Analysis Inc. Expression values were calculated using the Affymetrix GeneChip Operating System (GCOS) and were based on the difference of the PM (Perfect Match) signal and MM (Mismatch) signal at each of the probe pairs. The fluorescent signal values from each array were normalized to achieve similar overall intensity between arrays. The fluorescent signal values of any hybridization reaction were then multiplied by a scaling factor to make their (trimmed) mean intensity equal to 500. The scaling factor is unique to each hybridization reaction and is in general below 10
|Jun 28, 2007
|Last update date
|Aug 14, 2011
|NC State University
|42218 Gardner Hall
|Transcriptional and translational gene regulation in the maize pulvinus