Overview

The cDNA xProfiler compares gene expression between two pools of cDNA libraries, where each pool can be either a single library or several libraries. For a gene to be "present" in a library pool, there must be at least one EST sequence found in the UniGene cluster for that gene. The tool lists all the genes found in Pool A and Pool B and categorizes them as Unique and Non-Unique genes, then further as Known and Unknown genes. The results are finally tabulated to show how these genes are distributed between A and B.

What to Put In the Search Fields

Search Field	Options
1. Organism	Select "Homo sapiens" or "Mouse" from the drop down box.
2. Library Group	There are three options: Keep the default setting of "All EST Libraries" to search all cDNA libraries in dbEST. Select MGC libraries (The Mammalian Gene Collection) or CGAP libraries. Select both MGC and CGAP by highlighting one, holding down the [Ctrl] and [Alt keys] together, and highlighting the second one.
3. Minimum sequences	The minimum number of sequences per library is set at 1000 by default. However, a user may enter a lower minimum number to include libraries of interest with the understanding the significance of the results may be lowered.
4. List libraries by	Select any option to organize the list of libraries for review on the set-up page.
5. Pools A and B
i) Tissue Type	There are 4 options: The default setting is all tissues (nothing is highlighted) with the "Include" button selected. This searches all tissue types. Keep the default setting of all tissues, check "Exclude", and highlight one or more** tissues. This will search all the tissues in the list excluding the highlighted item(s). Select a single tissue and keep the "Include" button checked. Select two or more tissues and keep the "Include" button checked.
ii) Tissue Preparation	The default setting is all library preparation methods (described in Tissue Preparation Overview), or choose one specific method or several.
iii) Tissue Histology	The default setting is normal, pre-cancer, and cancer histology, or choose one specific histology or several.
iv) Library Protocol	The default setting is all library protocol methods, or choose one specific protocol or several. The protocols listed below the line are CGAP specific protocols described in cDNA Library Protocols Overview.
v) Library Name	With all of the above settings at default, enter the exact name of a CGAP or MGC library, e.g., NCI_CGAP_Pr1 or NIH_MGC_50.

** To choose multiple items in a select box, hold down the following keys together as you click on each item:

• In a PC: [CTRL] and [Alt]
• In a MAC: [Alt] and [Apple]

Examples of cDNA xProfiler Queries

The combination of different criteria for Pools A and B make the scope of possible gene expression comparisons range from simple to complex. Below are a few examples:

Pool A	Pool B
Human normal breast	Human breast cancer
Human brain cancer	Any human tissue, excluding brain
Human cell lines of prostate cancer	Human bulk prostate cancer
Normal human colon, normalized	Normal human colon, non-normalized
Mouse CGAP library NCI_CGAP_Mam6	Mouse CGAP mammary libraries

Reviewing Library Pools A and B

Having selected the criteria for Pools A and B, and pressed the Submit Query button, the page that next appears contains a list of the libraries that meet the chosen criteria.

• The first two columns of checkboxes sort which libraries belong to which pool.
• The next column provides the number of sequences in the library.
• The last column lists keywords that describe the library.

It may be necessary to resort the libraries, say to have all of Pool A followed by all of Pool B. To do this, click the back button on your browser and choose the appropriate criteria in #4. Press submit again.

Carefully review the libraries before proceeding. Check there are no libraries of "pooled" tissues or whole fetal tissue that may invalidate the results. Check that a library is not in both groups. Remove certain libraries to narrow your original selection. When you are satisfied, press Submit Query.

The cDNA xProfiler Results

The results page contains three sets of information:

• The UniGene Build number for this analysis. Since UniGene is rebuilt every two or three weeks, gene clusters may disappear or ESTs may be grouped in new clusters as more ESTs enter the database.
• Links to each set of libraries.
• The gene expression comparison results in tabular form.

As an example, here are the results of the comparison between all human normal and cancerous prostate tissue (without live links).

UniGene Build: Hs130 / Mm86 Libraries in A: 9 Libraries in B: 7 Pool Unique Genes Non-unique Genes   Known Unknown Known Unknown A 4 759 6789 5252 B 1 122 2317 1063 A or B 5 890 7047 5815 A and B 0 9 2059 482 A minus B 4 759 4730 4770 B minus A 1 22 258 581

The explanation of these numbers is as follows:

Pool	Unique Genes	Non-unique Genes
A	Genes found only in Pool A.	Genes found in Pool A and maybe in Pool B, and at least one other library.
B	Genes found only in Pool B.	Genes found in Pool B and maybe in Pool A, and at least one other library.
A or B	The total number of genes in Pool A plus Pool B. These genes are found in either A or B or maybe both, but not in any other library.	The total number of genes in Pool A plus Pool B. These genes are found in either A or B or maybe both, and in at least one other library.
A and B	Genes found in Pool A and Pool B, but not in any other library.	Genes found in Pool A and Pool B, and at least one other library.
A minus B	Equivalent to A unique	Genes found in Pool A and not in Pool B and also found in at least one other library.
B minus A	Equivalent to B unique	Genes found in Pool B and not in Pool A and also found in at least one other library.