RNA amplification and labeling was performed using the Ambion Amino Allyl MessageAmpTM II aRNA Amplification Kit as per the manufacturer’s protocol. In each amplification reaction, 1000ng of total RNA was used for starting material. The in vitro transcription reaction for synthesizing amplified RNA (aRNA) was performed for 12 hours. Probe aRNA was indirectly labeled by dye-coupling with Cy3 or Cy5 fluorescent dyes for hybridization to the cotton oligionucleotide gene chips.
Growth protocol
Cotton plants were grown in commericial field plots in Stoneville, MS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and an on-column DNase I digestion performed using the Sigma Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) as per the manufacturer’s protocol.
Label
Cy3
Label protocol
RNA amplification and labeling was performed using the Amino Allyl MessageAmpTM aRNA Amplification Kit (Ambion, Austin, TX USA) as per the manufacturer’s protocol. In each amplification reaction, 1000ng of total RNA was used for starting material. The in vitro transcription reaction for synthesizing amplified RNA (aRNA) was performed for 12 hours.
Hybridization protocol
For each aRNA probe labeled with either Cy5 or Cy3, 2ug was used for the hybridization. The slides were prehybridized 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 three times, followed by a final submersion in ddH2O 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.
Description
none
Data processing
Data quality was evaluated using the data distribution and ratio analysis tools. The data was normalized within arrays using Loess normalization and across arrays by median standardization (Dudoit et al., 2002). 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 using mixed models analysis (Wolfinger et al., 2001). The germplasm lines, developmental stage (DPA), the interaction between germplasm line and developmental stage, and dye channel were the fixed effects. The array effect was considered as a random effect.
Cotton Fiber Cells: Germplasm Line MD 52ne vs. Germplasm Line MD 90ne
Data table header descriptions
ID_REF
VALUE
background-subtracted median fluorescence intensities which have been normalized within arrays using Loess normalization and across arrays by median standardization
