Tutorial 1


Extend a set of microRNAs with regulatory information

CyTargetLinker provides a quick and extensive enrichment of biological networks in Cytoscape. The visualization options allow biological interpretation of complex regulatory networks in a graphical way.

In this tutorial a step-by-step explanation is given on how to extend a list of microRNAs with predicted and validated microRNA-target interactions (MTIs). (Note that in this tutorial the Cytoscape version 2.8.3 is used.)



First you will need to create a biological network in Cytoscape containing your microRNAs of interest. The datafile should contain at least two columns, (i) the microRNA name and (ii) the corresponding miRBase accession number. An example datafile, in which two microRNAs known to be involved in obesity are listed, can be found here. Open the datafile containing the microRNAs in Cytoscape via File Import Network from Table (Text/ MS Excel). In the panel you should select a file locally, select the column with microRNA names as the source interaction and transfer the header of the column as attribute names, as shown in Figure 1.

  • Figure 1: Import microRNAs into Cytoscape

Thereafter the miRBase accession number and possibly other characteristics of the microRNAs present as columns in the datafile used in step 1 should be added as attributes. Go to File Import Attributes from Table {Text/MS Excel). In the panel you should select a file locally, and transfer the header of the column as attribute names. The first column is automatically taken as the node identifier, as shown in Figure 2.

  • Figure 2: Add attributes to the microRNA network

The resulting biological network of two microRNAs from the example data, as shown in Figure 3.

  • Figure 3: microRNA network in Cytoscape



A regulatory interaction network (RIN) is a network containing regulatory interactions that are usually derived from an online interaction database. The networks are stored in XGMML (the eXtensible Graph Markup and Modeling Language)format, which is supported by Cytoscape. Each regulatory interaction consists of two nodes, a source (regulatory component) and target biomolecule, connected through one directed edge. Several RINs for different species and interaction types are provided.

However CyTargetLinker is not limited to the provided RINs. A user can also create their own RIN. A detailed description on how to create your own RIN will soon be available as a tutorial.

Before starting CyTargetLinker you need to download the RINs here and store them in a directory. The RINs provided on the CyTargetLinker website are species-specific. In this tutorial the human RINs are needed. Download all the RINs for homo sapiens, depicted in Figure 4.

  • Figure 4: Provided RINs for homo sapiens



Install the CyTargetLinker app via the plugin manager in Cytoscape version 2.8.3.

Open the CyTargetLinker in Cytoscape via Plugins CyTargetLinker plugin Load Regulatory Interaction Networks. Thereafter the CyTargetLinker selection panel is displayed (Figure 5).

  • Figure 5: CyTargetLinker selection panel

1. Select the user network. This is the biological network you want to extend with regulatory information. Here we select the human microRNAs.

2. Select the network attribute. This is the column containing the identifier which is supported by CyTargetLinker. The supported identifiers are listed in overview of the RINS here. For the microRNAs we recommend to use the miRBase accession numbers.

3. Select the directory containing the species specific regulatory interaction networks (RINs) which you downloaded in step 2.

4. Specify the name of the extended biological network. By default the plugin/app adds CLT_ to the name of the initial network.

5. Specify the direction of the interaction. Interactions from source to target (adding target nodes) or from target to source (adding regulatory nodes) or both (default) can be selected.

Click ok en thereafter select the RINs containing microRNA-target information (Figure 6).

  • Figure 6: RINs selection panel

The extended network is now visualized in a new Cytoscape network, as shown in Figure 7. To visualize the microRNA names and gene names in the network go to View Show Graphical Details. In the network microRNAs and target genes are defined as grey circles and pink rounded rectangles, respectively. The predicted MTIs are visualized in purple (TargetScan: 2556) and orange (microCosm: 1554) and the validated MTIs in blue (miRTarBase: 16) and red (miRecords: 6), as shown in the results panel on the right hand site. The color of the edges can be adapted in the results panel by click on the color. The hide&show and threshold functionalitites will be explained in step 4.

  • Figure 7: Extended microRNA network with MTIs



The visualization options in CyTartgetLinker make is possible to investigate the regulatory network in more detail.

Hide and show functionality

It is possible to hide or show specific RINs.

Here we are interested in only the validated MTIs. Go to the results panel and hide the RINs containing predicted MTI, i.e., TargetScan and MicroCosm, by clicking on the dropdown menu. Now only the RINs from miRTarbase and miRecords are shown in the network and the layout is immediately adapted (Figure 8).

  • Figure 8: Validated MTIs using hide&show functionality

Threshold functionality

It is possible to show interactions that are present in at least a specific number of RINs.

Here we are interested in MTIs that are present in at least 3 RINs. Go to the results panel, make sure all the RINs are shown, and select 3 RINs in the dropdown menu at supported by. Now the overlap between the RINs is shown and the layout is immediately adapted (Figure 9).

  • Figure 9: Overlap MTIs using threshold functionality