germplasm line: Texes Marker-1 (TM-1) developmental stage: -1 day of antheis (DOA) plant growth conditions: field tissue: unfertilized ovule treatment: none
Treatment protocol
The unfertilized ovules were placed in a liquid basal medium in the presence or absence of 5μM indole-3-acetic acid (IAA) and 1μM GA. Cotton ovules were incubated for 1, 3, 6, and 12 and harvested for RNA extraction.
Growth protocol
Cotton ovaries harvested from the cotton squares (-1 DPA) were surface-sterilized and dissected under sterile conditions. The unfertilized ovules were transferred to a liquid basal medium and incubated at 32oC in a 5% CO2 atmosphere.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Sigma Spectrum ™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO) with DNase1 digestion according to manufacturer's protocol.
Label
Cy5
Label protocol
RNA amplification and labeling using the Amino Allyl MessageAmp(TM) aRNA Amplification Kit (Ambion, Austin, TX) as per manufacturer's protocol. 1000ng of total RNA used for starting material of each amplification reaction. The in vitro transcription reaction for synthesizing amplified RNA (aRNA) performed for 12 hours.
Hybridization protocol
2ug aRNA probe labeled with either Cy3 or Cy5. The slides were pre-hybridized in 5X SSC, 0.1% SDS, and 0.1 mg/ml BSA (Fraction V) at 42oC for 45 minutes and then submerged in 0.1X SSC for 5 minutes 3X, followed by final submersion in ddH20 for 30 seconds. The slides were dried by centrifugation at 1000 X g for 5 minutes. The probe hybridization buffer consisted of 35% formamide, 5X SSC, 0.1% SDS, and 0.1 mg/ml sheared fish testes DNA. All hybridizations were carried out at 42oC for 16 hours in a Genetix 10-slide Hybridization Chamber (Genetix, New Milton, England). Following hybridization, slides were washed in 2X SSC, 0.1% SDS at 42oC for 5 minutes, then washed twice in 1X SSC at room temperature for 3 minutes, then washed twice in 0.1X SSC at room temperature for 2 minutes, and finally washed once in 0.05X SSC for 30 seconds. After drying by centrifugation, the slides were treated with DyeSaver2 (Genisphere Inc., Hatfield, PA, USA) as per the manufacturer’s protocol to prevent ozone-mediated degradation of the Cy5 dye.
Scan protocol
Slides were scanned in a GenePix 4000B microarray scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) at 10mm resolution using the GenePix Pro 6.0 software package.
Data processing
Data quality was evaluated using data distribution and ratio analysis tools. The data was normalized within and across arrays using Loess normalization (Dudoit et al., 2002; Wolfinger et al., 2001). The normalized expression levels were further evaluated for quality using correlation and principal components analyses. The normalized expression levels were then analyzed in individual gene-specific models. The gene-specific models were fit in JMP® Genomics 3.2 using mixed models analysis (Wolfinger et al., 2001). The hormone treatment, treatment time, the interaction between treatment and time, and dye channel were the fixed effects. The array effect was considered as a random effect.
