Index of Help
1. Query form
2. Illustration of gene plasticity (GPL)
3. Add new curve
4. Probe set quality
5. Electronic sorting
6. GSE mode for electronic sorting
7. GSM mode for electronic sorting

1. Query form

We recommend users use Internet Explorer8.0, Mozilla Firefox 21, or their higher versions to browse the database. ImmuSort can be accessed through Query Form (Figure 1) in the main page. In the current version, only human and mouse gene expression data derived from Affymetrix Human Genome U133 Plus 2.0 Array and Affymetrix Mouse Genome 430 2.0 Array respectively, are searchable. In the current version, there are 31 human and 17 mouse cell groups from 10,422 and 3,929 microarrays, respectively, and totally 20,283 human and 20,963 mouse genes are available to query the database. CCL20 is used as an example to navigate the database.
Fig.1
Figure 1. Query form. Please select the species and cell type before typing query text in the form of HUGO gene symbol or alias, Entrez GeneID, and probe set ID. To search human CCL20 (chemokine (C-C motif) ligand 20) in PMNs/neutrophils, user can type gene symbol, such as CCL20, SCYA20, MIP-3a, MIP3A, MIP-3-alpha (either in lower case or UPPER CASE) or gene ID "6364" for gene, or "205476_at" for probeset into the search box, then click the "Search" button.

2. Illustration of gene plasticity (GPL)

The result is shown in Figure 2. There are four sections in the result. Gene expression profile, which is directly reflected by a rank-based gene expression (RBE) curve, of queried gene or probe set across various experimental conditions is illustrated in Section 1. The x-axis represents the percentile rank scores ranging from 1 to 100, while the y-axis represents sample ratios at indicated rank score. Through the curve, the variability of gene expression, i.e., gene plasticity (GPL), is clearly illustrated. Generally, high plasticity is indicated by a wide-shaped RBE curve, while low plasticity is indicated by a narrow-shaped RBE curve. In Section 2, new curves or gene expression profiles can be added (see below), thus the comparison of GPL and gene expression of different genes can be made. Section 3 provides the link to electronic sorting based on gene expression intensity and the links to quality evaluation of the queried probe set (see below). GPL is measured by GPL score. In the ImmuSort database, the GPL score of a probe set is the difference of 3rd Qu. and 1st Qu. (i.e. GPL score = 3rd Qu.- 1st Qu.) of percentile rank scores of the probe set in all samples within the same cell group. Statistical parameters including the average rank score (mean), median, 1st Quartile (Qu.), 3rd Qu., and marker evaluation score (MES) are shown in Section 4.
Fig.2
Figure 2. Illustration of gene plasticity of CCL20 in PMNs/neutrophils.
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3. Add new curve

New curves can be added to compare the GPLs and gene expression profiles of different genes in the same cell group or of the same gene in different cell groups. To do so, user must choose a cell group and type a gene symbol, gene ID or probe set ID in the search box shown in Section 2 in Figure 2, and click the “Add” button. For example, as shown in Figure 3, when the RBE curve of CCL20 in the “B cell” group is added, the result indicates that CCL20 is highly plastic in PMNs while it has low plasticity and tends to be lowly expressed in B cells. The result also reveals that high expression levels are presented in a great portion of PMNs, of which the experiment conditions or sample sources can be retrieved by electronic sorting (see below). Currently, addition of new curves is restricted in the same organism. For visual consideration, the limitation of total additional curves is ten. However, if user uses the gene symbol or gene ID as search key words to add new curve, the curve counts may exceed five because some genes have more than one probe set.
Fig.3
Figure 3. Comparison of GPL and gene expression of CCL20 in PMNs and B cells.
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4. Probe set quality

When clicking "Blast" or "Blat" (Section 3, Figure 2) button, the target sequence of the probe set will be shown in a message box popped up. User can copy the sequence and make cross-hybridization analysis to confirm the reliability of the probe set. The tools, BLAST and BLAT can help the user to make sequence similarity (BLAST) and genome alignment (BLAT) analyses for the probe set, respectively. The button "Open Blast" or "Open Blat" in the message box links out to the web site of NCBI BLAST and UCSC BLAT, respectively.
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5. Electronic sorting

For wet-experiments during gene function studies, more attentions are often paid to that under what treatment conditions or disease states or in what cells, a gene is mostly up- or down-regulated. ImmuSort provides an important function to retrieve cell states or experimental conditions based on gene expression intensity. As shown in Section 3 of Figure 2, after finishing the selection of probe set and pressing “Show Detail”, the result of RBE curve will be shown in another mode, like Figure 4.
Fig.4
Figure 4. Electronic sorting can be mediated by the bins associated with rank scores.
The histogram facilitates interactive manipulation. The bins in Figure 4 provide links between sample information (including experimental conditions) and rank scores. Electronic sorting can be mediated by clicking the bin or selecting a bin range associated with interested rank scores after a display mode has been selected. For bin range selection, first click the button in the blue box at the top right corner of the page, then just click and hold the mouse cursor on a column in the left side and release it over another column. Currently, two display modes, GSE and GSM, are provided. When click the button in the green box in the top right corner of the page to restore the original histogram.
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6. GSE mode for electronic sorting

GSE mode provides a list of all Gene Expression Omnibus (GEO) series that contain the samples associated with each rank score. For example, if user clicks the bin "90" (Figure 4), the result shown in Figure 5 will be displayed, which comprises five GSEs. The right snapshots provide a quick view of differential gene expression in the samples of corresponding GSEs. User can click these snapshots, such as the second snapshot (GSE39889), to see more detailed information, including sample titles, signal values, rank scores, and detection p values (Figure 6).
Fig.5
Figure 5. GSE mode. There are five GSEs containing PMN samples with the rank score “90” for CCL20. The rightmost snapshots provide links to see more detailed information. When click the leftmost GSE name, a NCBI page of the corresponding GSE item will open in a new page.
Fig.6
Figure 6. Gene expression information of CCL20 in the PMN samples from GSE39889. The highlighted rows (in blue) indicate PMN samples with the rank score "90" for CCL20. When click the leftmost GSM name, a NCBI page of the GSM item will open in a new page. Only parts of PMN samples are shown.
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7. GSM mode for electronic sorting

GSM mode provides a list of all GEO samples (GSMs) associated with each rank score. For example, if user clicks the bin "90" (Figure 4), the result in Figure 7 that comprises six GSMs will be shown.
Fig.7
Figure 7. GSM mode. There are six GSMs with the rank score "90" for CCL20 in PMNs. When click on the GSM or GSE names, a NCBI page associated with the item will be opened in a new window.
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