ProteoMirExpress Help

1.        How to view a demo

 

Click “here” to see an instant sample result or to fill the sample data

If you fill the sample data into the input page, click “submit” to start a sample run:

Then you can enter the sample result from “My Jobs”:

You will see a finished sample test, then click “sample”

 

 

The inferred miRNA-centered regulatory network will be visualized on a Cytoscape page. The network contains regulatory relationships between miRNAs and their targets. Each node is a miRNA or a target gene, and each edge is an arrow pointing out from a miRNA or a TF to its target, which indicates the miRNA’s directly or indirectly suppressing regulatory function. The weights of the lines are proportional to the IS scores of the miRNA-target pairs. Targets from different expression gene classes are labeled in different colors. For instance, the class “UD”, which is colored as blue notes, represents genes that have mRNA levels unchanged (U) but protein levels decreased (D); while class “ML”, which is colored as light purple notes, represents genes with medium expression level in mRNA (M) but low expression level in protein (L).

On the second tap, the inferred active miRNAs are ranked according to their p-values in potential target gene set. When the users input miRNA expression data, the server will report two lists of significant miRNAs, one is listed in the “Inputted miRNA” tap which contains the miRNAs that have high expression or significant expression changes according to the inputted data, the other is in the “All enriched miRNA” tap which also includes other miRNAs that are not inputted miRNAs but have enriched target genes with expression changes. Users can select a sub-group of miRNAs of their interests from one or both miRNA lists to redraw the regulatory network. Edges between a miRNA from “Inputted miRNA” list and its targets will be shown as purple lines, while edges between other enriched miRNAs and their targets will be grey lines. Indirect targets are also shown in the network. Arrows pointing to an indirect target are in sage green. Click on a miRNA node, a list of its targets will pop up. Hovering over a target node, users can find its inputted identifier and gene symbol. Click on it, detail information of the gene will be found in its web page from National Center for Biotechnology Information (NCBI).

 

2.        How to start a new run

 

Open the data submit page by clicking on “Home”

 

 

 

(1)     Input your job title, which facilitates you to find your job in your job list (required).

 

(2)     Select the species your data from, only “Human” and “Mouse” are available.

 

(3)     Whether the input data is log2(fold change). When it is selected, all data of protein, mRNA and miRNA should be log2(fold change).

 

(4)     mRNA and protein expression data (required). You should firstly select the what kind of data you want to input, mRNA and protein in one file, mRNA and protein in two file, mRNA alone or protein alone.

mRNA and protein in one file should have three columns: the first is the IDs of the gene, the second is the expression (change) of mRNA and the third is the expression (change) of protein.

When the “No. of biological stage” (13) is 1, which means that your sample(s) is collected from one data point:

NM_006289 4563      32

NM_024329 7866      124

When the “No. of biological stage” (13) is 2, which means that your samples are collected from two data points before and after a certain treatment:

NM_006289 3.56       4.32

NM_024329 0.76       0.34

When the “No. of biological stage” (13) is 2 and “Using log2” (3) is selected:

NM_006289 1.83       2.11

NM_024329 -0.40      -1.55

P-value that represents the differentiation significance can also be taken into account. Then the input should be like:

NM_006289 1.83       0.01       2.11       0.02

NM_024329 -0.40      0.12       -1.55      0.35

 

mRNA and protein in two file: mRNA and protein data are separated in two file, each should have two columns, the format is the same as miRNA (4). The identifier of mRNA and protein can be different. ProteoMirExpress can recognize protein and mRNA identifiers from various databases, such as RefSeq, Ensemble, UCSC, Uniprot, PDB, etc. ProteoMirExpress will match the mRNA and protein automatically.

 

mRNA: when only mRNA data is available, use this option. The format is like:

When the “No. of biological stage” (13) is 1, which means that your sample(s) is collected from one data point, your input file should contain two Columns, the first is the ID of mRNA and the second is its expression level:

NM_006289 4563

NM_024329 7866

When the “No. of biological stage” (13) is 2, which means that your samples are collected from two data points before and after a certain treatment, your input file should contain two Columns, the first is the ID of mRNA and the second is the fold change of its expression levels:

NM_006289 3.56

NM_024329 0.76

When the “No. of biological stage” (13) is 2 and “Using log2” (3) is selected

NM_006289 1.83

NM_024329 -0.40

P-value that represents the differentiation significance can also be taken into account. Then the input should be like:

NM_006289 1.83       0.01

NM_024329 -0.40      0.12

 

Protein: when only protein data is available, use this option. The format is the same as mRNA above.

 

(5)     Input the data or upload the file (required).

 

(6)     The cutoff to classify mRNA.

For example, for one-stage sample, when mRNA expression cutoff is 0.33, it means that the mRNAs with expression level at the top 33% will be regarded as highly expressed mRNA, last 33% will be regarded as mRNA with low expressions. For two-stage sample, when the input is fold change of miRNA level between two stages, mRNA expression cutoff is 2, it means that mRNAs whose expressions increase more than two fold will be regarded as up-regulated mRNA, and mRNAs whose expression decrease more than one half will be regarded as down-regulated mRNA; when the input is log2(fold change), mRNA expression cutoff of 1 has the same effect as mRNA expression cutoff is 2 when inputting fold change

 

(7)     mRNA expression p-value cutoff.

P-value that represents the differentiation significance can also be taken into account when it is inputted like (4). If the p-value cutoff is set to be 0.05, for example, only genes whose significant p-value lower than 0.05 will be classified as significant increased or decreased genes.

 

(8)     Protein expression cutoff is similar to that of mRNA (6).

 

(9)     Protein expression p-value cutoff is similar to that of miRNA (7).

 

(10) miRNA expression data (optional) in the following format (columns should be separated by tab):

When the “No. of biological stage” (13) is 1, which means that your sample(s) is collected from one data point, your input file should contain two Columns, the first is the ID of miRNA and the second is its expression level:

hsa-miR-29c 24768

hsa-miR-224 13950

When the “No. of biological stage” (13) is 2, which means that your samples are collected from two data points before and after a certain treatment, your input file should contain two Columns, the first is the ID of miRNA and the second is the fold change of its expression levels (Stage with miRNA/Stage without miRNA):

hsa-miR-29c 2.35

hsa-miR-224 0.78

When the “No. of biological stage” (13) is 2 and “Using log2” (3) is selected

hsa-miR-29c 2.35

hsa-miR-224 -0.78

P-value that represents the differentiation significance can also be taken into account. Then the input should be like:

hsa-miR-29c 2.35       0.01

hsa-miR-224 -0.78      0.12

 

(11) The cutoff to classify miRNA:

For example, for one-stage sample, when miRNA expression cutoff is 0.33, it means that the miRNAs with expression level at the top 33% will be regarded as highly expressed miRNA, last 33% will be regarded as miRNA with low expressions. For two-stage sample, when the input is fold change of miRNA level between two stages, miRNA expression cutoff is 2, it means that miRNAs whose expressions increase more than two fold will be regarded as up-regulated miRNA, and miRNAs whose expression decrease more than one half will be regarded as down-regulated miRNA; when the input is log2(fold change), miRNA expression cutoff of 1 has the same effect as miRNA expression cutoff is 2 when inputting fold change

 

(12) miRNA expression p-value cutoff.

P-value that represents the differentiation significance can also be taken into account when it is inputted like (10). If the p-value cutoff is set to be 0.05, for example, only genes whose p-value lower than 0.05 will be classified as significant increased or decreased genes.

 

(13) No. of biological stage: “1” means that your sample(s) is collected from one data point, “2” means that your samples are collected from two data points before and after a certain treatment, which may affect what you should input into the web server (4,6,7,8,9,10,11,12)

 

(14) miRNA-mRNA interaction score: we define an Interaction Score (IS) to represent the reliability of the targeting relationship between a miRNA and its target as the proportion of the target databases containing the gene with at least one miRNA target site. We assign different weight for three different groups of databases according to their false positive levels (Equation 1).

9.jpg

where I = (TargetScan, miRanda, PicTar, PITA), J = (miRecords, TarBase). The users can input the cutoff to filter low score targets. For example, if cutoff is 0.5, then only targets reported in at least two of four computational predictions, or any one of CLIP/Degradome-seq database and experiment collection databases, will be selected for the analysis

 

(15) Statistical type. You can select to use hypergeometric test or permutation test to assess the significant of a miRNA whose targets overlap with the inputted potential suppressed genes.

 

(16) The statistical p-value cutoff, default is 0.05. Only miRNA whose targets significantly overlap with the inputted potential suppressed genes (gene class “All” in the gene “Type”) will be reported in the results (This p-value lower than the cutoff).

 

(18-21) Parameters for indirect target searching.

 

(17)  Promoter Range. The region of suppressed genes for searching the binding sites of miRNA-targeted transcription factor.

 

(18) TF Database. Select TF databases to get PWM for TFBS scanning (Multiple selection).

 

(19) PWM Scan P-value. The p-value cutoff for the significant of promoter to contain a TFBS.

 

(20) Conservation Filtering P-value. How significant that a TFBS is conserved between human and mouse.

 

(21) Submit your inputs

 

3.        How to get your results

 

When your job is finished, you will enter the result page:

In the tag of “Inputted miRNA Network” or “All Enriched miRNA Network” shows the miRNA centered regulatory network with the top significant miRNA and their targets. Yellow is miRNA, other colors represent different target classes (point to the icon  , you will see the color of each class). You can view the network in a larger window by click “” in the top-left corner. Network is available to be downloaded with the “” button. You can zoom in or out the network with the control panel “” in the bottom-right corner. You can move any notes. Click on it, detail information of the gene/miRNA will be found in its web page from National Center for Biotechnology Information (NCBI) or miRBase.

 

 

Click the tag of “Inputted miRNA” or “All Enriched miRNA”, you will get the results in table format. Potential targets are classified into three gene classes (different colors in the network): “DD” contains genes that both mRNA and protein are down-regulated, “UD” contains genes that mRNA levels are unchanged but protein levels are decreased, and “DU” are genes that mRNA levels are decreased but protein levels are unchanged. “LL” contains genes that both mRNA and protein have low expressions, “ML” contains genes that mRNA levels are medium but protein levels are low, and “LM” is genes that mRNA levels are low but protein levels are medium. Click on each type (gene class), you will get the list of miRNA that have targets in this type and click on the miRNA you will get the list of its targets in the type.

When the users input miRNA expression data, the server will report two lists of significant miRNAs, one is listed in the “Inputted miRNA” tap which contains the miRNAs that have high expression or significant expression changes according to the inputted data, the other is in the “All enriched miRNA” tap which also includes other miRNAs that are not inputted miRNAs but have enriched target genes with expression changes. Users can select a sub-group of miRNAs of their interests from one or both miRNA lists to redraw the regulatory network. Highlight the miRNA by clicking on the row, select multiple miRNA and then click “”. A new network will be shown in a pop-up window. Please click “” to check the miRNAs you have selected for network reconstruction. Because paging selection is supported by recording the selected miRNA into browser cookies, you may have some miRNAs you selected for previous jobs in the cookies. If so, click “”, and then click “” to clear the selected miRNAs recorded in cookies before you make any new selection to redraw the network.

Edges between a miRNA from “Inputted miRNA” list and its targets will be shown as purple lines, while edges between other enriched miRNAs and their targets will be grey lines. Indirect targets are also shown in the network. Arrows pointing to an indirect target are in sage green. Click on a miRNA node, a list of its targets will pop up. Hovering over a target node, users can find its inputted identifier and gene symbol. Click on it, detail information of the gene will be found in its web page from National Center for Biotechnology Information (NCBI). You can download the table with miRNA information, interaction information and indirect target information by clicking “”.

 

You can rank all record by click on the head of each column, for example rank by the “Hypergeometric P-value”. Click once sort by ascending order and click again sort by descending order.

You can search a particular miRNA by enter its name into the blank block under the head of “miRNA” column, and then “Enter”.

 

Detail information of targets and interactions can be visualized by click on a miRNA. A pop-up window will be shown. Again, you can search a particular item by entering it into the blank block in the second row of the table. In the link of interaction contains the detail information of miRNA binding sites from different data sources. The complementary binding site can be visualized click on the icon in “RNAHybrid” column.

 

4.        How to retrieve your previous jobs

 

On the left of the page, you have a list of jobs you run on the same computer. Your results will be stored in our server for a long time (a couple of months).

However your Internet Explorer may clear your cookie automatically when you close the web page. So please do remember to save the link of your important result by hovering over  and copying the link.

You can retrieve your result any time after it is finished.