MA Plot

MA Plot

DNA microarrays consist of an arrayed series of thousands of microscopic spots of DNA which allow comparisons between two samples of RNA or DNA (target samples), which can provide data on relative gene expression levels (in the case of RNA) or gene copy number (for DNA). An M plot is a mathematical tool that determines if there is a need to correct data for differences between microarray plates that are not due to the biological data itself. For example, if you were to go and look at actual microarray pictures you would see that some of the images may be brighter than the others. This difference is not due to the biological data but to differences in the experimental system that produced the images. Normalization corrects for this kind of difference without changing the biological data. All sorts of things can effect how a micro array works. Examples are changes in humidity between preparations or using a slightly smaller concentration of experimental material. Such small differences are virtually impossible to avoid. Fortunately we can correct for them across sets. Normalization is a "clean up" tool to reduce the background noise so you can see the real data. MA plots are used to determine if the data needs normalization and test if the normalization worked.

The data obtained from two-color DNA microarrays come in the form of fluorescent Red (Cy5) and Green (Cy3) dye intensities. One-color oligonucleotide arrays use only a single fluorescent dye.

Microarray data is often normalized within arrays to control for systematic biases in dye coupling and hybridization efficiencies, as well as other technical biases in the DNA probes and the print tip used to spot the array. By minimizing these systematic variations, true biological differences can be found. To determine whether normalization is needed, one can plot Cy5 (G) intensities against Cy3 (R) intensities and see whether the slope of the line is around 1. An improved method, which is basically a scaled, 45 degree rotation of the R vs. G plot is an MA-plot. The MA-plot is a plot of the distribution of the red/green intensity ratio ('M') plotted by the average intensity ('A'). M and A are defined by the following equations.

M is, therefore, the log intensity ratio (or difference between log intensities) and A is the average log intensity for a dot in the plot. MA plots are then used to visualize intensity-dependent ratio of raw microarray data (microarrays typically show a bias here, with higher A resulting in higher |M|, i.e. the brighter the spot the more likely an observed difference between sample and control). The MA plot uses M as the y-axis and A as the x-axis. The MA plot gives a quick overview of the distribution of the data. In many microarray gene expression experiments, the general assumption is that most of the genes would not see any change in their expression. Therefore the majority of the points on the y axis (M) would be located at 0, since Log(1) is 0. If this is not the case, then a normalization method such as LOESS should be applied to the data before statistical analysis. (On the diagram below see the red line running below the zero mark before normalization, it should be straight. Since it is not straight, the data should be normalized. After being normalized, the red line is straight on the zero line and shows as pink/black.)

The MA plot is the application of a Bland–Altman plot for visual representation of two channel DNA microarray gene expression data which has been transformed onto the M and A scale.