Microarray experiments
To measure the fluctuations in the signal intensities without taking gene expression into account, the images that were analysed were derived from several types of microarrays consisting of "self-to-self" hybridizations of different independent biological samples (for details see below and Table 1). To compare, within the array, the reproducibility of the ratios of the intensities between two biological conditions from one scan to the next, we carried out an experiment in which two different samples were hybridized (slide #5).
All the microarrays used in this study were pangenomic long-oligonucleotide (50–70 mers) arrays but they had different surface chemistries. Phosphoramidite arrays, commercial or custom, were manufactured by Agilent Technologies (Santa Clara, CA, USA). Hydrogel slides were distributed by an academic platform (French national microarray production site – CEA – Evry, France). Aminosilane microarrays were produced by an academic platform (University of Arizona, USA).
The biological samples were derived from various organisms (plant, fungus, yeast and Archea) (Table 1). Target preparation, hybridization and washing were done according to the manufacturers' instructions using the Gif/Orsay DNA Microarray platform's ISO 9001 protocols. The labelling was done via a linear amplification of antisense RNA with direct integration of CyDyes using the Low RNA Input Linear Amplification kit (LRILAK; Agilent Technologies). For some samples (Archea), single strand cDNA were synthesized, and then coupled with dyes using a Superscript™ Indirect cDNA labelling system (Invitrogen, Carlsbad, CA, USA). In each case, the labelling efficiency and product integrity were checked according to criteria defined by Graudens et al. [19]. Identical amounts of Cy3- and Cy5-labelled targets were mixed according to the manufacturer/distributor's instructions and incubated on the microarray slides for 17 hours at 60–65°C, in a rotating oven, using an Agilent hybridization system (Agilent technologies). The slides were washed and, then, any traces of water were removed by centrifugation at 800 rpm for 1 min or air dried with ozone-free dry air ("canned air").
Scanning and acquisition of images
The slides were scanned with an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) equipped with 532 and 635 nm excitation lasers for Cy3 and Cy5, respectively. This device allows acquisition of both images simultaneously and different modes of scanning: fixed or automatically adjusted settings, averaged line scanning (LS mode).
Each slide was scanned at 100% laser power at 5 and/or 10 microns resolution. All the scans were performed the same day, the number of successive scans (Nscans) in a series is, thus, limited to 8 per PMT setting. The procedure was as follows: Nscans at PMT voltage 400 V for Cy5 and 400 V for Cy3; Nscans at 500 V and 500 V; Nscans at 600 V and 600 V and finally Nscans at 700 V and 700 V. These settings are within the linearity range of the scanner.
For some slides (see Table 1), "AutoPMT" scanning was done, i.e. voltages were automatically adjusted to balance the distributions of the red and the green intensities and to optimize the dynamics of image quantification. The number of saturated pixels was low (less than 0.05%). These images were acquired using the "line scanning" mode (LS mode), e.g. each line of the array is scanned 1, 2 or 3 successive times before scanning the next line; from these multiple measurements an arithmetic mean is computed. This "AutoPMT" series was conducted as following: Nscans with each line scanned once; Nscans with each line scanned twice and averaged and Nscans with each line scanned three times and averaged.
Image registering and spot finding
From one scan to the next, the "red" or "green" images may be slightly shifted and, moreover, for each scan, the two images are not perfectly aligned. We, therefore, corrected the shift between the images by using the program for registering two-color microarray experiments described in the paper of Tang et al [10], allowing the sub-pixel adjustment of all the images of the series at the same time. The green image of the first scan was used as the reference for the alignment of all the green and the red images. The precision was 1/4 pixel for images obtained at 10 microns resolution and 1/8 pixel for those at 5 microns.
The images were analysed with the GenePix 6 software, the segmentation method being the adaptive circle method. In our experiments, the background was very low and was, thus, not subtracted. Not found, saturated and bad (dust, scratch, etc) spots were discarded.
Adjustment of fluorescence intensity
All the following analyses were carried out with the R statistical package available at http://cran.r-project.org/.
The median value of pixel intensities was used to quantify the spot intensity. It does not take the extreme values of pixels into account and, thus, it is the most common type of measurement. Within a series of successive scans having the same scanning parameters, spot intensities fluctuate. To compare the values of the median intensities obtained for each spot we adjusted them as follows:
, where F
s, n
is the median intensity (F635 or F532) of the spot, s, at scan number, n,
. n
spots
is the number of spots and med(
) is the median
value (n = 1: Nscans). This adjustment allows the comparison of images within a series of successive scans by homogenizing the mean signals
of all scans. The median of the mean signals of the n scans (med(
)) is used so that excessively divergent scans are not taken into account.
Quantification of the signal variability from one scan to the next
To evaluate the variability of the signal measurements between two successive scans we computed for each channel, (f), and for each spot, (s), a percentage of variation from one scan, (n), to the next, (n+1): 
When this percentage is larger than 10%, the spot was considered as an "outlier". We chose this threshold since it is a good compromise; it permits the detection of small fluctuations while still maintaining enough spots for a robust statistical study. Therefore, since we performed Nscans successive scans, each spot can be an "outlier" between 0 and (Nscans -1) times.
In order to study the variations of the signal from one scan to the next as a function of the spot intensity, the number of "outlier" spots was computed, separately, for successive intensity intervals. In theory, the log2 of the intensity varies from 0 to 16 but in practice it is never lower than 4.
Quantification of the instability of the intensity ratios within a series of scans
In microarray experiments the expression difference is usually quantified by the log2 intensity ratios (M values). To evaluate the instability of the M values within a series of scans, we computed e for "outlier" and "non-outlier" spots:
e is the average, for all the spots, of the larger of the absolute differences between the M values and their means within a series of scans.
The formula is valid for "outlier" and "non-outlier" spots, n
spots
being, on the one hand, the number of spots considered as being "outliers" and, on the other hand, the number of spots considered as being "non-outliers".
Analysis of intra-array probe repetitions
Some arrays used in this analysis contained replicated spots. These allow multiple measurements for the same probe in the same hybridization experiment. In yeast microarrarrays from Agilent technologies (slide #5), 2/3 of the probes are replicated and spotted randomly (see Table 1). For data coming from scans carried out using the "LS mode", the standard deviations of the
,
and of the M
s, n
values of the replicates for each probe were computed as a whole and, also, computed separately for each class of intensity and for M values as a function of the A value (A = 1/2(log2 (
) +log2 (
))).