Supporting Figure 1
Supporting Figure 2
Supporting information S1.
Materials and methods
Microarray protocol
For each of the four samples, 1 μg of cDNA was labeled with Cy3 or Cy5
dUTP/dCTP (Amersham Biosciences, Piscataway, NJ) followed by purification in a
single spin-column (Qiagen PCR purification kit, CA), and eluted in 25 μL of water.
Prior to hybridization, the microarray slides were 4X rehydrated at 50 °C on a
water bath for ten seconds and then snap-dried at 65 °C. Next, the slides were UV crosslinked (180 mJ) in a Stratalinker (Stratagene, CA). The slides were then incubated in a
1% BSA solution in 6.6× SSC at 37°C for 40 minutes, placed in 1% SDS for five min,
washed in water, and spun to dryness. The hybridization buffer was prepared using 24 μL
of labeled cDNA (including both the Cy3 and Cy5 samples), 1.2 μL of 2% SDS, 3 μL of
20× SSC, and 1.8 μL of Liquid Block (Amersham Life Science, cat# 1059304). The
samples were denatured for five minutes at 100 °C and placed immediately on ice. The
hybridization buffer (60 ul) was loaded onto each slide under a cover slip (Lifter slip,
Erie Scientific, 241x301-2-511) and incubated for 12-16 hours at 65 °C in a HYBAID
mini hybridization oven. After hybridization, the slides were washed successively in
three solutions (2× SSC and 0.5 % SDS at 65°C, 0.5× SSC at room temperature, and 0.2×
SSC at room temperature) for five minutes each. The slides were centrifuged to dryness
at 1,000 rpm for 2 minutes and scanned using a GenePix Autoloader 4200AL. The
scanned images were saved as individual, 16-bit grayscale, multi-image TIFF files. The
intensity values at each individual wavelength are therefore preserved for future analysis.
After data extraction, the results were saved as GPR (GenePix Results) or TXT doc. files.
Microarray data analysis
The GPR result file was analyzed in R software (R Core Development Team,
2008). A group of points was clearly up-regulated in the SVA1-rice relative to the wild
type, so the normalization procedures were designed to take this feature of the data into
consideration. The data were normalized within slides using a print-tip loess after
dropping points with greater than two-fold difference within a slide (approximately 6.3%
of points dropped) and no background adjustment (Smyth, 2005). The data were
normalized between slides within genotype (WT or transgenic) using the quantile method
(Smyth et al., 2003). For differential expression analysis, a mixed model ANOVA using
shrinkage estimators (Cui and Churchill, 2003) with genotype, environment, the
interaction of genotype and environment, and dye as fixed effects and array as a random
effect were fit (R/maanova; Cui and Churchill, 2003). Main effects of genotype and
environment and their interaction were considered significant with an adjusted p-value
(fdr; Storey, 2003) less than 0.05. Post-hoc pairwise contrasts were fit for significant
effects as appropriate, with subsequent fdr adjustment.
Reference:
Cui, X., and Churchill, G.A. (2003). Statistical tests for differential expression in cDNA
microarray experiments. Genome Biol. 4, 210.
Smyth, G.K. (2005) Limma: linear models for microarray data. In R Gentleman, V
Carey, S Dudoit, R Irizarry, W Huber (ed.), Bioinformatics and computational
biology solutions using R and bioconductor, Springer, New York, NY p. 397-420.
Smyth, G.K., Yang, Y.H., Speed, T. (2003) Statistical issues in cDNA microarray data
analysis. Methods Mol. Biol. 224, 111-136.
Storey, J.D. (2003) Statistical significance for genome wide studies. Proc. Natl. Acad.
Sci. USA. 100, 9440-9445.
Supporting Table S2. Primer sequences used for sq/qRT-PCR analysis of genes
Primer
OsVHAc1_Fwd
OsVHAc1_Rev
OsSORK_Fwd
OsSORK_Rev
OsLPD1_Fwd
OsLPD1_Rev
OsGPA1_Fwd
OsGPA1_Rev
OsPRBetV_ Fwd
OsPRBetV_ Rev
OsPlasto_ Fwd
OsPlasto_ Rev
OsACT1_Fwd
OsACT1_Rev
TR047684_Fwd
TR047684_Rev
OsMT1_Fwd
OsMT1_Rev
OsSCPL1_Fwd
OsSCPL1_Rev
OsAAA-ATPase_Fwd
OsAAA-ATPase_Rev
Sequence (5’-3’)
TCGGTATCTACGGCCTCATC
CTTTGGTTGCTGTGCATTTG
TGCTGCACTGAGAGGATACG
CTGCAGCAGTGGTGAGTTTC
ATGCAAGGTGACCCTGTACC
GGCGATGTACACCAGACTCC
AGCAGAGCAACACATCCACA
CAAAGCCAGTTTGGAAAAGG
GAGAGGCTGTGGAAGGTCTG
CACCCTGCTCTTAACCTCCA
CTGTGCTGGCTGAAACACAT
GCCCGGTTTGTAGTTGAAAA
TCCTCCGTGGAGAAGAGCTA
GCAATGCCAGGGAACATAGT
GCTACGGCGAGAACCTCTT
ACATCACCTGCGTGTAGTGG
TGAGAAGATCACCACCACCA
TTAGCAGTTGCAGGGGTTG
GCTACGGCGAGAACCTCTT
ACATCACCTGCGTGTAGTGG
ACATCTACGGGGTTGCAGTC
TAATGCCTGCGCTTACTCCT