Captured cells were homogenized in 500 µl of RNA extraction buffer (200 mM sodium borate decahydrate (pH 9), 30 mM ethylene glycol bis-N,N’-tetraacetic acid (EGTA), 1% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) sodium deoxycholate, 2% (w/v) polyvinylpyrrolidone (PVP), 0.5% (v/v) Nonidet-40 (NP-40), 10 mM dithiothreitol (DTT)). After 250 µl of ethanol was added to the captured cells, the sample was loaded onto a Qiagen RNeasy mini column (Qiagen, Valencia, CA) and washed as per the manufacturer’s recommendation.
Label
Cy5
Label protocol
RNA was amplified with a Amino Allyl MessageAmp™ aRNA amplification Kit (Ambion, Austin, TX). Two rounds of amplication were performed to get sufficient quantities of aRNA. Amplified RNA was coupled with Cy5 dyes (Amersham Biosciences, Piscataway, NJ) and purified using Qiagen RNeasy mini column (Qiagen, Germantown, MD). About 1 µg of fragmented Cy5-labeled aRNA probes was used for hybridization.
Captured cells were homogenized in 500 µl of RNA extraction buffer (200 mM sodium borate decahydrate (pH 9), 30 mM ethylene glycol bis-N,N’-tetraacetic acid (EGTA), 1% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) sodium deoxycholate, 2% (w/v) polyvinylpyrrolidone (PVP), 0.5% (v/v) Nonidet-40 (NP-40), 10 mM dithiothreitol (DTT)). After 250 µl of ethanol was added to the captured cells, the sample was loaded onto a Qiagen RNeasy mini column (Qiagen, Valencia, CA) and washed as per the manufacturer’s recommendation.
Label
Cy3
Label protocol
RNA was amplified with a Amino Allyl MessageAmp™ aRNA amplification Kit (Ambion, Austin, TX). Two rounds of amplication were performed to get sufficient quantities of aRNA. Amplified RNA was coupled with Cy3 dyes (Amersham Biosciences, Piscataway, NJ) and purified using Qiagen RNeasy mini column (Qiagen, Germantown, MD). About 1 µg of fragmented Cy3-labeled aRNA probes was used for hybridization.
Hybridization protocol
One Cy3-dCTP reaction is mixed with one Cy5-dCTP reaction to make one probe. Therefore, two “identical” probes each containing an equal amount of Cy3- and Cy5-labeled cDNAs were hybridized with two slides, which constituted one dye-swap experiment. The dye-swap was repeated once as a technical replication. The large dye-swap (four slides) was repeated using another biological sample (e.g., RNAs isolated from different pools of 3-DPA ovules). Therefore, each experiment consists of four technical replications and two biological replications in a total of eight slides (Chen et al. 2004). Hybridization was performed overnight (∼14 h) at 65°C. After hybridization, the slides were washed twice for 4 min each in 2X SSC, 0.2% SDS, again twice for 2 min each in 0.2XSSC, and twice for 2 min each in 0.05X SSC.
Scan protocol
The slides were scanned using GenePix 4000B (Axon, Foster City, CA), and the images were captured by GenePix Pro 4.1 software.
Description
Gossypium hirsutum L. cv. TM-1
Data processing
After the data were processed using log2 ratios of green and red hybridization signals, a robust and locally weighted linear regression (lowess) (Cleveland 1979) was used to remove non-linear components (e.g. dye and pin effects) (Quackenbush 2002). For the duplicate spots in each feature, we used an average value for data analysis. No additional steps for data normalization and background subtraction are needed for the AVONA model (Lee et al. 2004). The data were then subjected to the analysis of variance (ANOVA) test in a linear model to estimate the significant changes in gene expression caused by the two treatments (genotypes) (Black and Doerge 2002; Lee et al. 2004). A standard t-test statistic was used for this comparison based on the normality assumption for the residuals. The standard false discovery rate (FDR) (Hochberg and Tamhane 1987) was applied to control multiple testing errors using a significance level α= 0.05.
