1. (1) Normalization of cDNA microarray data Methods, Vol. 31, no. 4, December 2003 Gordon K. Smyth and Terry Speed

2. Normalization for two-color cDNA microarray data The purpose of normalization is to adjust for effects which arise from variation in the microarray techno- logy rather than from biological differences between the RNA samples or between the printed probes. Variation in the microarray technology 1. within array: The dye-bias generally vary with intensity and spatial position on the slide. 2. between array: Differences may arise from differences in print quality, ambient conditions when the plates were processed or simply from changes in the scanner settings.

3. M-A plot Write R and G for the background-corrected red and green intensities for each spot. The log-ratios of expression, M = log 2 R - log 2 G. The log-intensity of each spot, A = (log 2 R + log 2 G)/2, a measure of the overall brightness of the spot.

4. Diagnostic plots  dye-bias and i ntensity : MA-plot (Fig. 1)  Spatial variation : Spatial image plot (Fig. 2) Boxplots of the M-values for each print tip group (Fig. 3)  Combining Spatial and i ntensity trends: Individual loess curves for each print tip groups (Fig. 4)

5. Fig. 1. MA-plot for an array showing three different trend lines. Blue : shows the median of the M-values. Orange : shows the overall trend line as estimated by loess regression. Yellow : shows the loess curve through a set of control spots known to be not differentially expressed.

6. Fig. 2. Spatial plot of green (cy3) back- ground values. The array was printed using a 12 x 4 pattern of print tips. The background tends to be more intense: around the edges in the four corners in tip rows 8 and 9 and columns 3 and 4

7. Fig. 3. Boxplots of the M-values for each print tip group. The M-values are higher : in the middle of each sequence of four and in the middle of the overall sequence (tip rows 7, 8, and 9).

8. Fig. 4. Individual loess curves for the 48 print tip groups. For this array, the slope and shape of the curves is broadly consistent over the print-tip groups although the height varies. The height of the curves varies between tip groups in a similar way to the height of the boxplots in Fig. 3.

9. Global loess

10. Print-tip loess normalization We recommend this method as a routine normalization method for cDNA arrays. It corrects the M-values both for sub-array spatial variation and for intensity-based trends. Print-tip loess

11. Two-dimensional loess We do not use this method as a routine normalization strategy because of  concern that imperfections on the array may present sudden rather than smooth changes and  concern that the two-dimensional loess curve may confuse local clusters of differential expression on the array with the spatial trend to be removed. Another way to model spatial variation is to use a two-dimensional loess curve. This can be combined with intensity-based loess normalization to give the two- dimensional normalization strategy,

12. Composite normalization may be used when a suitable set of control spots is available which are known to be not differentially expressed. Control spots: 1. To be of most use in loess normalization, the control spots should span as wide a range of intensities as possible. 2. A satisfactory set of controls for this purpose is a specially designed microarray sample pool (MSP) titration series in which the entire clone library is pooled and then titrated at a series of different concentrations. Composite loess normalization

13. Yang et al. (2002) propose the composite normalization

14. Correcting for other trends Print-order effect ( Fig. 5 ) the numerical order in which the spots were laid down during the printing of the array

15. Fig. 5. Plate or print-order effects for the first slide in the ApoAI knock-out experiment report- ed by Callow et al (2000). This array was printed with a 4 x 4 arrangement of print- tips and with 19 rows and 21 columns in each tip group. This means that the print-order index goes from 1 to 19 x 21 = 399 and that 4 x 4 =16 spots share each print-order index. The plot shows that a series of plates starting around print- order 169 have higher median M-values than the rest of the array. Indeed, it turns out that spots with print orders between 169 and 252 were printed with DNA from a different library to the other spots. median M-value 169 252

16. One can normalize for this print-order effect by subtracting from the M-values the medians shown in the plot. One would then proceed on to print-tip loess normalization. Print-order normalization

17. Weighting for spot quality Most image analysis programs routinely record a variety of descriptive information about each spot. If this information is used to construct a numeric quality measure for each spot, then lower quality spots can be down-weighted in the normalization process. Spot quality 1. spot size 2. roundness of the spot 3. background intensity 4. SNR 5. foreground or background regions 6. spot location

18. A more comprehensive measure of spot quality: weighting spots according to spot area (the number of pixels in the segmented foreground region of the spot) Inspection of the TIFF images of arrays used in the examples suggests that the area in pixels of an ideal circular spot on these arrays is about 165 pixels. 165 330 (=2*165) The values from the weight function are used as relative weights in all the loess regre- ssions used in the normal- izations.

19. This weight function is a simple function of only one of the morphological characteristics of the spot and more complex quality measures can easily be imagined. Other measures of spot quality computed from the image analysis output could be used in the same way to provide weights in the normalization.