Programa de Doctorado en Biotecnologa Universidad de Santiago de Chile Facultad de Qumica y Biologa Depto.

de Ciencias Biolgicas

LAB II TRANSCRIPTOMIC - MICROARRAY Tpicos avanzados de bioinformtica y biotecnologa aplicada

Dr. Rodrigo Vidal

Molecular Biology research evolves through the development of the technologies used for carrying them out. It is not possible to research on a large number of genes using traditional methods. DNA Microarray is one such technology which enables the researchers to investigate and address issues which were once thought to be non traceable. One can analyze the expression of many genes in a single reaction quickly and in an efficient manner. DNA Microarray technology has empowered the scientific community to understand the fundamental aspects underlining the growth and development of life as well as to explore the genetic causes of anomalies occurring in the functioning of the human body. A typical microarray experiment involves the hybridization of an mRNA molecule to the the DNA template from which it is originated. Many DNA samples are used to construct an array. The amount of mRNA bound to each site on the array indicates the expression level of the various genes. This number may run in thousands. All the data is collected and a profile is generated for gene expression in the cell.

PART I 1.- Download the file FILE 1.txt and open it with Excel 2.- Select the sheet Data which contains the red and green intensities corresponding to 16 mice, 8 control and 8 with a gene knocked out. 3.- Perform a scatterplot of Red vs Green for mouse 1 columns 2 and 3. Are the points scattered around a straight line with slope 1? 4.- Artifacts can be seen better using M-A plots. Download the file FILE 2.txt and open it with Excel.

5.- Do an M-A plot (selecting the appropriate colums for mouse 1). Are the points symetrically scattered around the x-axis?. CAN YOU REPRODUCE THE SAME DATA?.

PART II The box-plot is a very good tool to perform quality assessment. It can be used to compare experiments (1 box-plot per mouse) or to compare expression groups inside the array (1 box plot per printing tip).

1.- Open the file FILE 3.txt with some statistical package. 2.- To perform a boxplot for multiple experiments go 3.-Select all control columns (columns 3 to 10) and perform the graph. What do you see?

PART III 1.- Donwload and Open TMeV (http://www.tm4.org/mev/) select as file type "Stanford" from the file type menu. 2.- Open the file "FILE IV.txt". To finish loading you must select the upper leftmost expression value and click the Load button. 3.- Select T-Test from the main menu 4.- Define the groups you want to compare by assigning each column to group A (Control) or B (Knocked out mice) 5.- Choose the procedure for calculating p-values (t distribution), the desired significance level (0.05) and an adjustment for multiple testing (adjusted bonferroni) When you press OK, a series of folders appear on the left window. Explore them to see in graphical and numerical form, which genes have been selected as differentially expressed. Write down their ID and look at the gene description in the original file. Does this gene seem to be part of lipid metabolism?

PART IV

TIGR MeV provides a variety of clustering methods. In addition, many distance or similarity metrics are proposed (although the choice of the metric is ignored for some algorithms, see the manual for precisions).

Open TMeV and create a new multiple experiment viewer Select as file type "Stanford" from the file type menu. Open the file "FILE V.txt". To finish loading you must select the upper leftmost expression value and click the Load button.

Notice that this file has already been normalized. You should thus avoid to use the normalization functions from MeV.

Hierarchical clustering
We will start by applying Hierarchical Clustering using the Pearson Uncentered distance

In the Distance menu, select Pearson Uncentered. Open the Analysis menu and select the HCL function from the list or press the HCL button. Select Complete linkage and leave "Cluster genes" and "Cluster Experiments" selected

A new item "HCL (1)" appeared in the tree displayed in the left panel. Click on the handle to expand this item. Click on HCL Tree. The tree resulting from hierarchical clustering appears in the right panel. We will change its display properties to obtain a global view of the tree.

Select the command Display/Element size/Other, and specify a height of 3 pixels and a width of 10. Right-click on the tree. A contextual menu appears. o In this menu, select the command Gene tree properties, o Set the minimum pixel height to 6, and click Apply dimensions. This changes the size of the tree branches, to better emphasize the different hierarchical levels. By clicking on any node of the tree, you select all its descendent branches. Select a node which contains genes with similar profiles of expression. Noticed that the rest of the tree is now displayed in vanishing colors. Select a node of interest (for example a node whose descendent have aparently the same expression profiles). Right-click on this node. A contextual menu appears. o Select Store cluster. This allows you to assign a name, a description (comments) and a specific color to this cluster. The command File - Save image allows you to save the tree image in various formats. Support trees