Will my GE probes work on the Agilent platform?

If you have Expression type probes, especially ones that have been designed outside of eArray, you can use the GE Probe Check tool to see if those probes will work on the Agilent platform, with Agilent protocols. You can compare probes to a given transcriptome, and get an analysis of potential probe targets and possible cross-hybridization problems. You can check a probe group that is already in the eArray system, as well as probes in a file that you upload.

Note: GE Probe Check is not available to guests, or within a collaboration.

Before you check GE probes

You must be an eArray registered user.
You must have a TDT or FASTA format file of probe sequences, or have a probe group within eArray that contains the probes of interest. Prepare a compressed *.zip format version of the file for upload purposes.
(Optional) Prepare a FASTA format transcriptome file for eArray to use to evaluate potential targets and cross-hybridization problems. Prepare a compressed *.zip version of the fie for upload purposes. Note: Transcriptomes for many species are already available within eArray.

To check GE probes

Click the Workspace tab.

Set the application type to Expression.

Click Probe > GE Probe Check.

The GE Probe Check page appears.

Set the following parameters. All are required.

Parameter	Instructions/Details
Probe Details
Design Job Name	Type a name that will later enable you to identify this GE Probe Check job.
Species	Select a species. This is the species that is associated with the probes to be checked.
Select one of these options	Upload Probe File – Select this option to check the probes in a file you upload. Click Browse. A dialog box appears. Select the desired TDT format or FASTA format probe data file, then click Open. The location of the file appears in Upload a Probe File.
Select one of these options	Select a Probe Group – Select this option to check the probes in an existing probe group. To select one or more probe groups, click Select and Add. See Select probe groups for probe searches.
Transcriptome Details
Select one of these options	None – Select this option if you do not wish to specify a transcriptome. If you choose this option, eArray checks only the base composition of your probes, and does not identify potential probe targets or cross-hybridizations.
	Select Agilent-Provided Transcriptome (by Species) – Select this option to use a pre-defined species transcriptome as a similarity database. Select this option if your target species is represented by one of the available transcriptomes. Select the desired species from the list.
	Upload Transcriptome File – Select this option to use an uploaded transcriptome data file as a similarity database. Click Browse. A dialog box appears. Select the desired FASTA format transcriptome file, then click Open. The location of your transcriptome file appears in Upload Transcriptome File.

Click Submit.

A message informs you that your job has been submitted, and that you will receive an e-mail when it is complete.
Click Exit.

Your job appears in the Search Results pane at the bottom of the GE Probe Check page, where you can monitor its status. The job status progresses from In Queue, to Designing, and when the job finishes, to Completed. If your job has a status of ERROR, eArray was not able to process it, and you must submit it again. eArray does not automatically update the page. Click Refresh to update the page to its latest version.

You may need to wait one day or more for your job to finish, depending on the number of jobs ahead of yours in the queue, and the amount of data submitted. When eArray finishes your job, it sends you an e-mail.
When your GE Probe Check job is complete, return to the GE Probe Check page.
In the Actions column of the Search Results pane, next to the desired job, click View Design.

The Probe Check Details page appears in a new window.
The table below describes the two types of results that are available. Click the corresponding tab at the top of the page to view each.

Result

Description

Probe Check Summary

General information and statistics about the set of probes that were examined by GE Probe Check.

Probe Check Details

The list of probes examined by GE Probe Check, with the sequence and base composition statistics of each probe, the accession number of the predicted target for each probe, and an analysis of potential cross-hybridization problems.

These results regarding predicted hybridizations may be of particular interest:

Predicted Target – This is the transcript in the similarity database that forms the most energetically favorable duplex with the probe sequence.

X-Hyb Potential – If you are trying to measure the expression level of a transcript, you must have probes that bind uniquely to this target transcript. If sequences other than the target transcript hybridize to a probe, this is a cross-hybridization (X-Hyb) event. The X-Hyb potential score is 0 if the only transcript predicted to form a duplex with a probe is the predicted target. If one or more other transcripts are predicted to form a duplex with a probe, the X-Hyb potential score is 1. A score of 0 is desirable.

X-Hyb Target – This is the transcript in the similarity database that forms the second most energetically favorable duplex with the probe sequence.

Click Close.

You can download the results and create a probe group based on the results. You can also delete the job.