Flowers were tagged on the day of anthesis (0 days post anthesis (dpa)). Developing bolls from the first fruiting positions closest to the stem were collected at 8 dpa between 8 a.m. and 10 a.m. to eliminate diurnal effects. Bolls were collected from different plants, and immediately mingled, frozen in liquid Nitrogen, and then stored in 50-ml polypropylene tubes at -80°C.
Growth protocol
G. barbadense L. cv. Pima S7, also known as Egyptian cotton, was grown in the green house under a 30/21°C day/night temperature regime in a randomized block design
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the modified hot borate method (Wan and Wilkins, 1994; Wilkins and Smart, 1996; Arpat et al., 2004)
Label
Alexa Fluor® 555 and Alexa Fluor® 647 (Dye Swap included)
Label protocol
Total RNA samples were reverse transcribed and fluorescently labeled using the amino-allyl reverse transcription labeling protocol (Hughes et al., 2001; Arpat et al., 2004). Total RNA (10 μg) spiked with 1000 pg of a reference mRNA spike mix (Lucidea Universal Scorecard, Amersham Pharmacia) was used to synthesize first-strand cDNA in the presence of amino allyl-modified dUTP (aa-dUTP) (Arpat et al., 2004, Hughes et al., 2001). The reverse transcription reaction mix contained ~10 µg “spiked” total RNA, 5g poly-T21VQ primers (Operon), 0.2 mM dNTPs (Sigma), 0.2 mM aminoallyl-dUTP/dUTP (Sigma) in a 2:3 ratio, and 2.5 U SuperScript III (Invitrogen). Following incubation for 2 hours at 45°C, the aminoallyl-labeled cDNA was purified using Microcon YM-30 columns (MilliPore) according to manufacturer’s instructions. Aminoallyl-cDNA was labeled using Alexa Fluor 555 and 647 succinimidyl ester dyes according to manufacturer’s instructions (Molecular Probes, Inc.), and purified using the QiaQuick PCR purification kit (Qiagen).
Channel 2
Source name
Developing Cotton fiber at 5 days post anthesis (dpa)
Flowers were tagged on the day of anthesis (0 days post anthesis (dpa)). Developing bolls from the first fruiting positions closest to the stem were collected at 8 dpa between 8 a.m. and 10 a.m. to eliminate diurnal effects. Bolls were collected from different plants, and immediately mingled, frozen in liquid Nitrogen, and then stored in 50-ml polypropylene tubes at -80°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the modified hot borate method (Wan and Wilkins, 1994; Wilkins and Smart, 1996; Arpat et al., 2004)
Label
Alexa Fluor® 555 and Alexa Fluor® 647 (Dye Swap included)
Label protocol
Total RNA samples were reverse transcribed and fluorescently labeled using the amino-allyl reverse transcription labeling protocol (Hughes et al., 2001; Arpat et al., 2004). Total RNA (10 μg) spiked with 1000 pg of a reference mRNA spike mix (Lucidea Universal Scorecard, Amersham Pharmacia) was used to synthesize first-strand cDNA in the presence of amino allyl-modified dUTP (aa-dUTP) (Arpat et al., 2004, Hughes et al., 2001). The reverse transcription reaction mix contained ~10 µg “spiked” total RNA, 5g poly-T21VQ primers (Operon), 0.2 mM dNTPs (Sigma), 0.2 mM aminoallyl-dUTP/dUTP (Sigma) in a 2:3 ratio, and 2.5 U SuperScript III (Invitrogen). Following incubation for 2 hours at 45°C, the aminoallyl-labeled cDNA was purified using Microcon YM-30 columns (MilliPore) according to manufacturer’s instructions. Aminoallyl-cDNA was labeled using Alexa Fluor 555 and 647 succinimidyl ester dyes according to manufacturer’s instructions (Molecular Probes, Inc.), and purified using the QiaQuick PCR purification kit (Qiagen).
Hybridization protocol
The hybridization was performed according to Arpat et al., 2004. Printed slides were pre-hybridized in 5X SSC, 0.1% SDS, and 1 mg/µl BSA in a sealed coplin jar for 45 minutes at 42˚C, followed by two washes of 10 sec each in filtered ddH2O preheated to 42˚C, and dried with filtered compressed air. Alexa-labeled cDNA (500 ng/fluor or the equivalent of 40 to 45 pmol of dye depending upon the frequency of incorporation (FOI)) was suspended in 45µl of hybridization buffer (25% formamide, 4.16X SSC, 0.08% SDS, and 0.1 µg/µl sheared salmon sperm DNA) and loaded underneath the cover-slip (M Series LifterSlip, 22x60mm, Erie Scientific) using capillary action. Following hybridization at 42ºC for 16-18 hours in a humidified hybridization chamber (Genetix), slides were washed sequentially at 42ºC in 2X SSC, 0.2% SDS for 5 min, 0.5X SSC for 3 minutes, 4 times in 0.1X SSC for 1 minute per wash, and 0.05X SSC for 10 seconds, followed by drying with filtered compressed air.
Scan protocol
Hybridized slides were scanned using a GenPix3000 array scanner (Axon Instruments, CA, USA) followed by feature extraction to quantify signal intensities using ImaGene 4.2 software (BioDiscovery).
Description
PDF files of detailed experimental protocols for RNA isolation (SOP MA002 v1.1), amino-allyl labeling (SOP MA003 v2.1) and microarray hybridization (SOP MA004 v2.1) can be accessed at Cotton Functional Genomics Center website.
Data processing
Data were filtered and corrected for bias using the following steps: (1) Low quality spots (dust particles, scratches, high background, or irregularly shaped spots) were manually flagged and excluded. (2) Spot intensities were corrected by subtracting the mean of the background intensities from the signal intensity to reduce background noise. (3) Corrected spot intensities lower than the mean of corrected buffer spot plus 3 STDEV (Standard Deviation) were excluded (Hüser et al., 2003). (4) The absolute ratios (M) of dye swaps were correlated and the upper most 30% quartile of divergent data points were removed. This cut-off value enhanced the correlation between dye swaps by increasing the linearity. (5) Exclusion of all genes in which <60% of their data points did not pass all filtration steps. Reference calibration spots, which were spiked-in at a 1:1 ratio in both channels, were weighted to 10 before normalizing data. In order to adjust for effects that arise from variation inherent to microarray technology rather than biological differences in the RNA samples, filtered data was normalized using intensity-dependent normalization (Yang et al., 2002). The robust scatter plot smoother ‘Lowess’ implemented in R (version2.0.1) statistical software package and LIMMA (version 1.9.0) was used to perform a local per chip normalization. To make full use of within-array duplicated spots, LIMMA’s pooled correlation method (Symth et al., 2003) was used to estimate the strength of the correlation between duplicated spots by fitting separate linear models to the expression data for each gene, but with a common value for the between-replicate correlations. Normalized expression data at each time point was used as a reference point for all other time points (global reference) to calculate the relative expression, meaning that the expression at any given time point will be estimated independently six times relative to each of the other six time points.
