Flower buds from 35S::AP3-GR, ap3-3, 35S::PI transgenic plants, DEX treated, collected at 0hr.
Extracted molecule
total RNA
Extraction protocol
Flower buds were snap frozen in liquid nitrogen. Total RNA was extracted using the Trizol (GibcoBRL, Fredrick, MD, USA) reagent and protocol. RNA was cleaned using the Qiagen RNeasy Mini Kit (GmbH, Hamburg, Germany).
Label
biotin
Label protocol
Single stranded cDNA is synthesized by reverse transcription using the poly (A) RNA present in the starting total RNA sample. Single stranded cDNA is then converted into double stranded cDNA and purified using the Affymetrix Cleanup Module. An in vitro transcription (IVT) reaction is then carried out overnight in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded cDNA. (information from keck.med.yale.edu/affymetrix/analysis.html)
Hybridization protocol
cRNA is fragmented in the presence of heat and Mg+2 before hybridization to the array. The sample is hybridized to the standard array for 16 hours at 45°C. The standard array is then washed and stained using the fluidics station and then scanned. (information from the Affymetrix Gene Expression website of the W.M. Keck Foundation Biotechnology Resource Laboratory)
Scan protocol
The scanner is a 16-bit sophisticated opto-mechanical confocal scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays. The scanner uses a high-quality, solid state 532nm Diode-Pumped, Frequency Doubled Nd:YAG (Neodymium-doped Yttrium Aluminum Garnet) Green Laser. The scanner optics are designed to condition the laser beam to deliver an excitation spot size of 3.5 μm and can detect as few as 400 phycoerythrin molecules in a 20 x 20 μm probe site. (information from the Affymetrix Gene Expression website of the W.M. Keck Foundation Biotechnology Resource Laboratory)
Description
0hr DEX Replicate 2
Data processing
The computer workstation with GeneChip Operating software controls the fluidics station and the scanner. GeneChip Operating software can control up to eight fluidics stations using preprogrammed hybridization, wash, and stain protocols for the probe array. The software also acquires and converts hybridization intensity data into a presence/absence call for each gene using appropriate algorithms. Finally, the software detects changes in gene expression between experiments by comparison analysis and formats the output into .txt files which can be used with other software programs for further data analysis. (information from the Affymetrix Gene Expression website of the W.M. Keck Foundation Biotechnology Resource Laboratory)
