Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction

Abstract

Background: High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.

Results: All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.

Conclusions: It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Figure 1

Mean spot intensity versus PMT voltage for three scanners of two different brands, ScanArray4000 I (A), ScanArray4000 II (B), and GenePix4000B (C). Three representative spots are presented, one with high intensities (●, ○), one with intermediate intensities (■, □), and one with low intensities (▲, △). The intensities of the green and red channel are represented by closed and open symbols, respectively. The PMT voltage is given in % of maximum voltage for ScanArray4000 and in voltage for GenePix4000B.

Figure 2

Mean spot intensity in a scan with saturation (scan 1) versus mean spot intensities in a scan of the same arrays without saturation (scan 2), obtained at lower PMT voltage. ScanArray4000 I was used during scanning. (A), (B), and (C) Data of the red channel. (D), (E), and (F) Data of the green channel. (- - - - -) The intensity levels of 50 000 and 200. The PMT voltages (% of maximum voltage) used in scan 1 and scan 2 were 56% and 51% (A), 59% and 52% (B), 65% and 58% (C), 63% and 56% (D), 65% and 57% (E), 67% and 57% (F).

Figure 3

Mean spot intensity in a scan with saturation (scan 1) versus mean spot intensities in a scan of the same arrays without saturation (scan 2), obtained at lower PMT voltage. The high intensity data of Figure 2 are shown (●) in addition to intensities corrected for saturation (○). ScanArray4000 I was used during scanning. (A), (B), and (C) Data of the red channel. (D), (E), and (F) Data of the green channel. (- - - -) The intensity level of 50 000. (…….) The intensity levels of 30 000 and 20 000. The PMT voltages (% of maximum voltage) used in scan 1 and scan 2 were 56% and 51% (A), 59% and 52% (B), 65% and 58% (C), 63% and 56% (D), 65% and 57% (E), 67% and 57% (F).

Figure 4

Normalized expression ratio (log₂(Norm/G), where Norm is the total intensity normalization factor) versus average intensity in the red (R) and green (G) channel (1/2(log₂G+log₂R)) plotted on a double logarithmic scale. ScanArray4000 I was used during scanning. The PMT voltage is indicated for the red and green channel separately (% of maximum voltage). The same data set without and with correction of saturated intensities are shown in (A) and (B), respectively. (C), (E), (G), and (I) The intensities in the green channel were above 200 (PMT voltage of 62%), whereas those in the red channel were to an increasing extent below this level (PMT voltages ranging from 56% to 40%). (D), (F), (H), and (J) The intensities in the red channel were above 200 (PMT voltage of 62%), whereas those in the green channel were to an increasing extent below this level (PMT voltages ranging from 56% to 40%). The discontinuity in the distributions at the lowest voltages was due to the apparent discrete values of the lowest intensities and standard deviations, showing values of 1, 2, 3, etc., which occurred because integers were used when assigning the values.