# Supplemental Data I 

#  Wheat 8K cDNA microarray fabrication and technical properties

Microarrays provide the opportunity to simultaneously monitor the
expression of thousands of genes at various developmental stages, thus
providing an opportunity to analyze the temporal patterns of expression
of genes in the same plant material grown in the same environmental
conditions. To conduct a comprehensive study of gene expression during

caryopsis development, we generated a cDNA microarray with 10,800 spots
representing 7,833 unique genes from wheat. About 30% of the unique
probes on the array were identified from cDNA libraries made exclusively
from mRNA isolated from developing endosperm and caryopsis tissue.
Furthermore, about 75% of the genes on the array have been mapped on the
hexaploid wheat genome.

Included on the array are different types of controls that can be used
for normalization, and data quality assessment: 18 AFGC microarray
controls, which include ten spiking controls, derived from human genes,
that do not show cross hybridization with plant RNA, and 23 Lucidea
ScoreCard (from Amersham Biosciences) calibration, ratio, utility and
negative controls which are artificial genes composed of sequences from
yeast intergenic regions. To assess the overall performance of the wheat
cDNA array, we analyzed the sensitivity and dynamic range of expression,
as well as the *within-* and *between-slide* reproducibility.

[*Sensitivity and dynamic range*.]{.underline} Analysis of the spot
signals of the 10 Lucidea Score Card calibration controls (*Figure A*)
shows that we can reliably detect a signal from 0.3 pg of the
calibration control RNA for every 1 μg of total RNA labeled for
hybridization. The data collected had a linear dynamic range of 10^4^.

Assuming that the amplification efficiency of the endogenous wheat RNA
is similar to that of the spiked-in calibration controls, the
calibration control data will allow us to approximate the number of
transcripts of each gene per ug of RNA used for hybridization. Using the
estimated 100-500 pg of total RNA in a plant cell (Goldberg et al. 1978;
Kerk et al. 2003), and an average of 1,500 nucleotides/molecule of plant
mRNA (Alexandrov et al. 2006; Goldberg et al. 1978; Ogihara et al.
2004), the cDNA microarrays are estimated to detect the expression of
genes with as low as 36 to 181 RNA transcripts per cell. Using RNA Cot
analysis, Goldberg and colleagues, estimated that there are 17
transcripts, 340 transcripts and 4,500 transcripts per cell for low,
moderate and highly expressed genes, respectively. Thus, cDNA arrays can
almost detect even genes with low level of expression.

*[Within-slide reproducibility]{.underline}.* To assess the
reproducibility of the probe signals within slides, we used the data
obtained from 10 AFGC mammalian spiking controls spotted on 45 of the 48
subgrids on the array. Each spiking control was spotted 45 times giving
450 data-points per slide per dye. Equal amounts of in vitro transcribed
mRNA of each spiking control were added to 10 ug total RNA before
indirect labeling with fluorescent dyes. The ratio of the intensity
signals for the spiking controls between the Red (Alexa 647) and Green
(Alexa 555) channels was expected to be 1. *Figure B* shows the
*within-slide* reproducibility of the spiking control spot signals
obtained from 9 slides. The different colors represent the signal ratio
from different slides. The coefficient of correlation between each
comparison ranges from 0.94 to 0.99, with an average of 0.97.

*[Between-slide reproducibility]{.underline}*. The *between-slide*
reproducibility was assessed both on technical and biological
replicates. To evaluate the reproducibility of technical replicates, the
probe signals from 9 slides used to hybridize RNA from one set of
biological samples were compared. *Figure C* shows an example of the
distribution of the spots from RNA from the same sample that was
independently labeled and hybridized to two different slides. Comparison
of the signals obtained from the two independently labeled RNA from the
same sample gave a coefficient of correlation of 0.95. Analysis of the
between-slide reproducibility of technical replicates on all 9 slides,
or 27 comparisons (data not shown) revealed a range of coefficient of
correlation of 0.85 to 0.96, with an average of 0.91. Similarly,
analysis of the between-slide reproducibility of biological replicates
(RNA isolated from 7DPA grains from different plants grown for this
experiment) on 27 slides, revealed a coefficient of correlation ranging
from 0.72 to 0.97 with an average 0.87. *Figure D* shows an example of a
comparison between two biological replicates that has high
*between-slide* reproducibility.

# 

# Figure: Wheat 8K cDNA microarray technical properties

![](media/image1.png){width="6.272222222222222in"
height="3.5618055555555554in"}

***A. Calibration curve from the ten Lucidea Universal ScoreCard
controls***

DNA of the 10 Lucidea Score Card calibration controls was spotted 4
times on each slide. In vitro transcribed mRNA for each calibration
control was spiked in different concentrations: 0.1, 0.3, 1, 3, 10, 30,
100, 300, 1000 and 3000 pg per 1 ug sample total RNA used before
labeling. Equal amounts of the calibration control RNA were added to
every sample RNA used for the experiment. Probe signals from both the
Red (Alexa 647) and Green (Alexa 555) channels from 9 slides were
analyzed. The average signal was calculated for each calibration
control. The signal intensities for the Red Alexa 647) and Green (Alexa
555) channels have been background subtracted. The average of the
corrected raw signal intensities (y-axis) were plotted against the
amount of the spiked-in RNA of the calibration control per 1 ug of
sample RNA labeled. The half bar on each data point represents the
standard deviation from the mean of the corrected signal for each
calibration control.

## **B. Spiking controls and within slide reproducibility**

Comparison of the Red (x-axis) and Green (y-axis) channel intensity
values for each mammalian spiking control spot within an array. The
different colors represent spots from different slides analyzed.

## **C. Between slide reproducibility --technical replicates**

Comparison of the intensity values for the same microarray spot on two
different arrays hybridized with the same RNA sample that was
independently labeled.

## **D. Between slide reproducibility --biological replicates**

Comparison of the intensity values for the same microarray spot on two
different arrays each hybridized with two distinct biological RNA
samples for the same time-point.