Table of Contents

• What is the Gene Expression Notebook (GEN)?
• GEN system requirements
• Using this document
• GEN terminology
• Downloading and installing GEN
• Opening GEN
• Selecting an action from the Main menu
• GEN assay sheets and worksheets
• Using worksheets
• Entering worksheet data
• Saving worksheet data
• Defining worksheet fields
• Genes (Genes)
• Probes (Probes)
• Probe Preparation Methods (Probe Prep)
• Immunohistochemistry and In Situ Specimens (Specimens)
• Immunohistochemistry and In Situ Specimen Prep (Specimen Prep)
• Antibodies (Antibodies)
• Antigens (Antigens)
• Primary Antibody Detection Methods (Primary Detection)
• Primer Pairs (Primer Pair)
• cDNA Generation Methods (CDNA Generation)
• PCR Conditions (PCR Conditions)
• Northern Samples (Northern Samples)
• Western Samples (Western Samples)
• RT-PCR Samples (PCR Samples)
• Creating new worksheet fields
• Using assay sheets
• Creating a new assay sheet
• Opening assay sheets
• Adding experimental details to assay sheets
• Using a Manage window to enter experimental details
• Adding specimens/samples
• Storing additional experimental details
• Storing results
• Recording results for in situ and immunohistochemistry assays
• Recording results for blot assays
• Copying information into a new workbook
• Determining standard nomenclature for mouse genes and alleles
• Validation in the GEN
• Submitting data to GXD
• Customizing GEN to your needs
• Troubleshooting the GEN
• Getting help
• Getting started using the GEN (tutorial)

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What is the Gene Expression Notebook?

The Gene Expression Notebook (GEN) is a modified Microsoft ® Excel ™ workbook for organizing and storing experimental data. Using GEN, you can:

• store the details, results, and images of data from experiments
• reuse previously entered reagents, specimens, and methods to define new experiments
• copy reagents, specimens, and methods into new notebooks so you don't have to reenter them
• create a central record-keeping resource for your laboratory reagents
• submit your mouse data for inclusion in the Gene Expression Database (GXD).

Because GEN is a species-neutral tool, you can use it to manage expression data from any organism. You can also customize it to include fields for details you consider important such as dates, freezer locations of probes or specimens, or any other record details.

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GEN system requirements

GEN runs in Microsoft Excel. The following are the version and memory requirements for optimum performance.

Macintosh Requirements
Versions	Excel 2004, Excel X
Operating systems	OS X, OS 9
Memory	The number of images and amount of experimental data GEN can hold is limited by the amount of memory assigned to GEN. Therefore, for Mac OS 9, set the Excel memory to at least 256 megabytes. See Troubleshooting the GEN for instructions on how to manually allocate more memory. Mac OS X will allocate memory automatically.
PC Requirements
Versions	Excel 2003, Excel 2000
Operating systems	Windows XP, Windows 2000
Memory	A Windows-based system should adjust the memory automatically.
Other Requirements
Macros	For GEN to operate properly, the macros must be enabled. This requires that, if given a choice, the Macro Security be set to Medium (or lower).
Updates/service packs	To ensure reliable operation of GEN, you should install all the latest updates and patches for your version of Excel. To make sure that your computer has all the necessary updates, go to http://office.microsoft.com/officeupdate/default.aspx and click on "Check for Updates."
Notes	GEN does not operate in Excel 2007 (for PCs) or Excel 2008 (for Macs). Although we no longer test GEN on older versions of Excel, we have no reason to believe it will not work on Excel 97 and Excel 98.

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Using this document

If you:

• are familiar with Microsoft Excel, you'll find the workbook, worksheet, and Manage window concepts easy to understand. See the Table of Contents for spreadsheet activities specific to the GEN application.
• are new to spreadsheets or to the GEN, GEN Terminology provides a list of GEN concepts.
• want to read about GEN from "soup-to-nuts," start with the Table of Contents and pick a topic.
• need to install GEN, see GEN system requirements.
• have GEN installed and are ready to plunge right in, try Getting started using the GEN (tutorial).
• want to modify the notebook forms for your own use, you'll find instructions for adding fields, changing tab names, and deleting data at Customizing GEN to your needs.

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GEN terminology

assay sheet	A modified Microsoft Excel spreadsheet that stores experimental results and images. The parameters of an experiment are defined on the assay sheet using worksheet data. The GEN has assay sheets for RNA In-Situ, Immunohistochemistry, Northern, Western, and RT-PCR experiments.
collapse	An operation done on assay sheets to hide rows containing experimental details in order to save screen space.
column	The vertical arrangement of cells on a GEN worksheet or assay sheet. Columns are identified by alphabetical headings: A, B, C, D, E, etc. The horizontal arrangement is called a row; rows are identified by numerical headings: 1, 2, 3, 4, 5, etc.
command button	Rectangular button that carries out a command. GEN has four on the Main menu: Create New Assay Sheet, Create New Submission, Validate this Workbook, and Copy info to new Workbook.
Copy info to new Workbook	Main menu command. When you click this button, GEN copies all worksheet data from the current notebook into a new notebook. It does not copy experimental data stored in any assay sheets.
Create New Assay Sheet	Main menu command. Used in conjunction with the Assay Type drop-down menu to create an assay sheet for storing experimental results.
Create New Submission	Main menu command. When you click this button, GEN allows to select the assay sheets of experiments you want to submit to GXD and prepares a file suitable for submission.
default	A setting or action assumed by a computer program when none is specified by the user. For example, on GEN's Immunohistochemistry assay sheet, the default view shows the antibody, but not antigen, details.
drop-down list arrow	Indicates a drop-down menu. You can select a desired value by scrolling the menu.
drop-down menu	A drop-down list arrow to the right of a field indicates a drop-down menu. You can select a desired value by scrolling the menu. If there is no drop-down menu associated with a field, you must type the desired value into it.
Validation Report	A worksheet in the GEN used in the Validation process. The validation function in the GEN checks for omissions in worksheet fields needed for submission of data to GXD. These omissions are then listed on the Validation Report, along with the worksheet or assay sheet on which they occur.
expand	An operation done on assay sheets to display rows containing experimental details.
field	An item of information such as a strain name, the Theiler stage, or the notes for an experiment. A required field is one in which you must enter data, while an optional field is one you may leave blank. A collection of fields is called a record.
GXD	Gene Expression Database. A community database that integrates different types of gene expression information from the mouse and provides a searchable index of published experiments on gene expression during embryonic development. GXD is part of the Mouse Genome Informatics (MGI) resource.
ID	The text in the first column of each worksheet is the identifier for that row. Each data record in the GEN must have an ID that is a unique name or number.
Main menu	Appears when you open GEN. Contains basic information and four command buttons for performing notebook functions: Create New Assay Sheet, Create New Submission, Validate this Workbook, and Copy info to new Workbook.
Manage window	A pop-up window that allows you to create or modify worksheet entries from an assay sheet without leaving that sheet. Accessed by clicking on a Manage button.
navigation tabs	Tabs appear along the bottom of notebook pages. Clicking a tab opens that notebook page. GEN adds a tab every time you create a new assay sheet. Not all of the tabs are visible at once. Either click the |<, <, >, >| arrows to scroll through the tabs or right-click (control-click for a single button mouse) on the arrows to open a window listing all the pages.
notebook/workbook	These terms are used interchangeably to refer to the Gene Expression Notebook.
notes	There are three types of Notes fields in the GEN. Notes on worksheets Assay Notes (on all assay sheets) Results Notes (on all blot assay sheets). Use the Notes field to record information that is important to know but that does not fit into any of the other assay or worksheet fields.
record	A collection of fields; for example, the contents of a GEN worksheet.
Results menu	Used on assay sheets. Allows you to add fields to the results section to help you organize the recording of your expression results.
row	The horizontal arrangement of cells on a GEN worksheet or assay sheet. Rows are identified by numerical headings: 1, 2, 3, 4, 5, etc. The vertical arrangement is called a column; columns are identified by alphabetical headings: A, B, C, D, E, etc.
specimens/samples	Assay sheet results are directly associated with specific specimens or samples. RNA in situ and immunohistochemistry assays use specimens. Blot assays use samples. You can include multiple specimens or samples in an assay sheet.
submission	This function allows you to select the assay sheets you want to submit to GXD. GEN takes this set of assays and makes a notebook containing the designated experimental data and any worksheet parameters used in these assays. You can then send this file to GXD for inclusion in the database.
validation	GEN checks to see if an entry contains the field information required for submission to GXD. You can validate a worksheet, an assay sheet, or the entire notebook. See Validation in the GEN.
Validate this Workbook	Main menu command button. Click to validate the entire workbook.
view	There are several ways sections of an assay sheet can be displayed: expanded, intermediate, and collapsed view. The expanded view displays all the data from the worksheet used for that section. The collapsed view displays only the ID. You toggle between them by clicking the >  button. The <  button is present only for Antibodies.
worksheet	A Microsoft Excel spreadsheet designed to hold specific types of data used to define an experiment. The following is a list of the GEN worksheets. The GEN tab name for the worksheet, if different, appears in parentheses. Genes Probes Probe Preparation Methods (Probe Prep) Immunohistochemistry and In-Situ Specimens (Specimens) Immunohistochemistry and In-Situ Specimen Prep (Specimen Prep) Antibodies Antigens Primary Antibody Detection Methods (Primary Detection) Primer Pairs (Primer Pair) cDNA Generation Methods (CDNA Generation) PCR Conditions Northern Samples Western Samples RT-PCR Samples (PCR Samples)

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Downloading and installing GEN

You can download GEN from the Gene Expression Notebook Resource Page. You can also access The Mouse Anatomical Dictionary here, as well as access this Help document. When you download GEN, you'll have to provide your name, e-mail address, and computer system type. We use this information to keep track of users so we can let you know about new releases and features. We do not share your information with anyone.

To download GEN:

1. Click on the phrase (Download the) Gene Expression Notebook.
2. Fill out the registration form and click Submit.
3. Click on Download the GEN. What happens next varies. In general:
   • With Excel 2004 and Excel X, GEN will be saved to the hard drive.
   • With Excel 2003, GEN will be saved to the hard drive, and the application will open.
   • With Excel 2000, you will be asked, "What do you want to do with this file?"
     Choose Save it to disk, and then type in a file name and a location to save it to.
4. You must Enable Macros to allow the GEN to operate optimally. Since GEN macros are unsigned, with some versions of Excel, you may need to lower the Macro Security level to Medium (or lower, if desired). To do this, go to the Tools menu and choose Options. Click on the Security tab and open the Macro Security dialog button to set the new security level.

You can now open the notebook.

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Opening GEN

Once you download GEN, there are two ways to open the application:

1. Double-click the GEN icon on your desktop.
2. Open Excel and click File—>Open and browse to locate the GEN.xls file.
3. Click Enable Macros when the prompt asks if you want to disable macros or enable macros. The GEN Main menu appears.

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Selecting an action from the Main menu

From the Main menu, you can:

• Access the Gene Expression Database (GXD) at Mouse Genome Informatics (MGI)
• Click the Help Sheet link to get more information.
• Use the command buttons to:

  Create New Assay Sheet	Generate new assay sheets to store your experimental results.
  Create New Submission	Make a file containing data selected for submission to GXD.
  Validate this Notebook	Check the notebook to ensure that all fields required by GXD contain entries.
  Copy info to new Workbook	Create a new notebook containing all the worksheet data from the current notebook.

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GEN assay sheets and worksheets

In the GEN, you store experimental details in worksheets and experimental results in assay sheets. The following table indicates which worksheets are used by each type of assay sheet.

Assay Sheet:	RNA In Situ	Immunohistochemistry	Northern	Western	RT-PCR
Worksheet:	Genes	Genes	Genes	Genes	PCR Samples
Worksheet:	Probes	Antibodies	Probes	Antibodies	Genes
Worksheet:	Probe Prep	Antigens	Probe Prep	Antigens	Primer Pair
Worksheet:	Specimens	Primary Detection	Northern Samples	Primary Detection	cDNA Generation
Worksheet:	Specimen Prep	Specimens		Western Samples	PCR Conditions
Worksheet:		Specimen Prep

Using worksheets

Worksheets store information on your experimental reagents and protocols. Assay sheets use worksheet entries to define the parameters of an experiment.

To open a worksheet, click the appropriate tab on the bottom of the worksheet.

• Worksheets contain fields. (You might be more familiar with the Excel term, cell). Fields hold data. The type of data to enter is identified at the top of each column. (The Probes worksheet, for example, contains the following fields: Probe Name, Gene, Sequence Type, Species, Sequence Acc #, Subsequence, Region Covered and Notes).
• Some fields list possible selections on a drop-down menu. To see if a drop-down menu is available on a given field, click in the field. If a drop-down list arrow appears, click it to activate the list. Scroll the list, and click your selection. In most cases, if the value you need is not listed, you can type it directly into the field.
• The first field in a worksheet is always the ID, the unique name or identifier for the reagent or method. A drop-down menu is generated from the first field of each worksheet. Therefore, this field cannot be left blank, and each entry within it must be unique. If you enter a previously used ID, a warning appears when you leave the worksheet.
• We hope we have designed fields that are of use for you, but use the fields in the notebook as you see fit. If there are fields you regularly use, feel free to add them as columns to the appropriate worksheets.
Although these fields will not be displayed on the assay sheet, they are copied when you create new notebooks. (See Creating new worksheet fields.)
• You can edit a worksheet by changing the data in any field and saving the notebook. Note, however, that changes are propagated to all assays using that record. A warning appears when you use a Manage window to change data in a worksheet.
• You must make your entries on consecutive rows. Skipping rows results in an error message when you leave that worksheet. A worksheet entry separated by blank rows will not appear in any drop-down menus.
• The blue headings of each worksheet link to the Help Sheet.

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Entering worksheet data

1. There are two ways to enter data into a worksheet:
   • Type text directly into the fields on the worksheet.
   • Use the Manage window on an assay sheet.
2. Type the identifier for this information in the first field. Remember that you must make this ID unique.
3. Continue filling out the worksheet by entering the appropriate data in each cell of a row.

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Saving worksheet data

There are several ways to save your data from the worksheet:

• Click the Save icon on the toolbar.
• Click the Excel File drop-down menu and select Save (File—>Save).
• Click File—>Exit, and when the prompt asks if you want to save your changes, click Yes. (If you use this method, you will also exit the program).

Note: We recommend that you save frequently when using the Gene Expression Notebook. If Microsoft Excel (or any other application) locks up, you'll lose any changes you have made to GEN.

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Defining worksheet fields

Titles below list the worksheet name first, followed by the tab name in parentheses.

Genes (Genes)

Assays used in:	RNA In Situ; Immunohistochemistry; Northern; Western; RT-PCR

Note: You must enter genes used for RT-PCR assays directly onto this worksheet. There is no Manage Genes window on the RT-PCR assay sheet to use for entering the information.

Field	Description
Name	Type the name of the gene whose expression is being analyzed. For tips on determining the official gene name in mouse, see Determining standard nomenclature for mouse genes and alleles.
Symbol	Type the symbol for the gene whose expression is being analyzed.
Notes	Enter any additional information you want stored with this record.

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Probes (Probes)

Assays used in:	RNA In Situ; Northern

Field	Description
Probe Name	Type a unique name for the probe ID
Gene	Type the name of the gene that the probe was derived from. (In most cases, this is the same as the gene being assayed.)
Sequence Type	Use the drop-down menu to specify whether the probe used is cDNA, genomic, oligonucleotide, or mitochondrial.
Species	Type the name of the species the probe was isolated from.
Sequence Acc #	If the probe corresponds to a sequence or fragment from a sequence in GenBank, type the accession number.
Subsequence	If applicable, specify the nucleotide range (with respect to the accession number above) that defines the probe.
Region Covered	Type the region of the gene that the probe covers (e.g., 3' UTR, homeobox domain, etc.).
Notes	Type any additional information you want stored with this record.

Note:  If you are planning on submitting data to GXD, we encourage you to provide as much sequence information about the probes used in your experiments as possible. Knowing the sequence of a probe enables integration of expression results with other information in the database that are related to the sequence.

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Probe Preparation Methods (Probe Prep)

Assays used in:	RNA In Situ; Northern

Probe Prep Method	Type a unique identifier for the probe preparation method.
Type	Use the drop-down menu to specify whether a DNA probe or riboprobe was prepared.
Hybridization	Use the drop-down menu to specify if the probe is antisense, double-stranded, or sense.
Labelled With	Use the drop-down menu to specify the label used.
Label Coverage	Use the drop-down menu to specify where the label was incorporated into the probe.
Visualized With	Use the drop-down menu to enter the method used to detect the probe.
Notes	Type any additional information you want stored with this record.

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Immunohistochemistry and In Situ Specimens (Specimens)

Assays used in:	RNA In Situ; Immunohistochemistry

Specimen ID	Type a unique identifier for the specimen.
Strain	Type the specimen strain. If the specimen is from mouse, see Inbred Strains of Mice and Rats for a listing of approved strain names.
Mutations	Type the allele description or symbol for the mutated gene. For tips on determining the official allele symbol in mouse, see Determining standard nomenclature for mouse genes and alleles.
Theiler Stage	If the specimen is from mouse, you can use the staging criteria of Theiler to define its age. If you do not know the Theiler stage, fill in the next three boxes. (You can leave them blank if you enter the Theiler stage.)
Age Unit	Use the drop-down menu to specify the age unit.
Age Value	Type the number that specifies the age along with the Age Unit. Leave this field blank if you specify either postnatal or adult as the Age Unit.
Noon of Plug Day=	If using embryonic rodent specimens, use the drop-down menu to specify whether the noon on the day of plug detection is defined in your lab as embryonic day 0, 0.5, or 1. Unknown is also an option. This allows embryo staging consistency in GXD.
Sex	Use the drop-down menu to specify the gender of the specimen, if known.
Notes	Type any additional information you want stored with this record.

Note:   If you are planning on submitting data to GXD, we require that you specify the specimen age (using either Theiler stages or age units).

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Immunohistochemistry and In Situ Specimen Prep (Specimen Prep)

Assays used in:	RNA In Situ; Immunohistochemistry

Spec Prep Method	Type a name or other unique identifier for the preparation method.
Type	Use the drop-down menu to specify section, whole mount, optical section taken from a specimen hybridized as a whole mount, or section taken from an embryo hybridized as a whole mount.
Fixation	Use the drop-down menu to specify the method used to fix the specimen.
Embedding	Use the drop-down menu to specify the method used to embed the specimen if sectioned. For whole mounts, leave this field blank or select Not applicable.
Notes	Type any additional information you want stored with this record.

Note: If you are planning on submitting data to GXD, we require that you specify the specimen type.

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Antibodies (Antibodies)

Assays used in:	Immunohistochemistry; Western

Note:  Since you need an antigen to complete an antibody record, it is easier if you enter the antigen information on the Antigens worksheet before using the Antibodies worksheet.

Antibody Name	Type the name or other unique identifier of the antibody.
Antigen Name	Type (or use the drop-down menu to specify) the gene name of the antigen used to generate the antibody.
Ab Generated In	Use the drop-down menu to specify the species the antibody was generated in.
Type	Use the drop-down menu to identify whether the antibody is monoclonal or polyclonal (or unknown).
Class	Use the drop-down menu to identify the immunoglobulin class of the antibody.
Functions In	Use the drop-down menu to identify the types of assays the antibody can be used in: immunohistochemistry, immunoprecipitation, Western blots, or any combination thereof.
Reference	Type the citation for the published reference describing the antibody or the name of the supplier.
Notes	Type any additional information you want stored with this record.

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Antigens (Antigens)

Assays used in:	Immunohistochemistry; Western

Name	Type the name of the antigen.
Sequence Acc #	If the antigen corresponds to a sequence or fragment from a sequence in GenBank, type the accession number.
Subsequence	If possible, specify the amino acid range (with respect to the accession number above) that defines the antigen.
Region Covered	Specify the region of the protein that was used as an antigen (e.g., homeobox domain, entire, etc).
Species	Specify the species the antigen was derived from, if applicable.
Strain	Type the specimen strain. If the antigen was derived from mouse, see Inbred Strains of Mice and Rats for a listing of approved strain names.
Tissue	Type the tissue the antigen was derived from, if applicable.
Theiler Stage	If the antigen was derived from mouse, you can use the staging criteria of Theiler to define its age.
Age Unit	If applicable, use the drop-down menu to specify the age unit of the antigen source.
Age Value	Type the number that specifies the age along with the Age Unit. Leave this field blank if you specify either Postnatal or Adult as the Age Unit.
Sex	Use the drop-down menu to specify the gender of the antigen source, if known.
Cell Line	Specify the cell line the antigen was derived from, if applicable.
Antigen Notes	Type any additional information you want stored with this record.

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Primary Antibody Detection Methods (Primary Detection)

Assays used in:	Immunohistochemistry; Western

Detection Method	Type the name of the primary antibody detection method.
Secondary Antibody	Type the name of the secondary antibody used.
Labelled With	Use the drop-down menu to enter the label used on the secondary antibody.
Notes	Type any additional information you want stored with this record.

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Primer Pairs (Primer Pair)

Assays used in:	RT-PCR

Note:  You must enter information to be stored in this worksheet directly. There is no Manage Primer Pair window on the RT-PCR assay sheet to use for entering the information.

Primer Pair Name	Type the name of the primer pair.
Gene	Type (or use the drop-down menu) to specify the gene whose sequence was used to generate the primers.
5' Sequence	Type the sequence of the 5' probe.
3' Sequence	Type the sequence of the 3' probe.
Expected Band Size	Type the expected size of the reaction product.
Sequence Acc #	If the primer sequence is derived from a sequence in GenBank, type the accession number.
Notes	Type any additional information you want stored with this record.

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cDNA Generation Methods (CDNA Generation)

Assays used in:	RT-PCR

Note:  You must enter information into this worksheet directly. There is no Manage cDNA Generation window on the RT-PCR assay sheet to use for entering the information.

cDNA Generation Method	Type the name of the method used for generating the cDNA.
Primer	Type the name or sequence of the primer used for cDNA synthesis.
RNA Type	Use the drop-down menu to specify the type of RNA used for the synthesis reaction
Amount Value	Type the amount of the RNA used in the cDNA synthesis reaction.
Amount Units	Use the drop-down menu to specify the unit used to measure the RNA amount.
Transcriptase	Type the name of the transcriptase used for cDNA synthesis.
Buffer	Type the name of the buffer used in the cDNA synthesis reaction.
Notes	Type any additional information you want stored with this record.

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PCR Conditions (PCR Conditions)

Assays used in:	RT-PCR

Note:  You must enter information into this worksheet directly. There is no Manage PCR Conditions window on the RT-PCR assay sheet to use for entering the information.

PCR Conditions Name	Type the name of the PCR conditions.
cDNA Amount	Type the amount of cDNA used in the PCR reaction.
Annealing Temperature	Type the temperature used for the annealing step in the PCR reaction.
Annealing Time	Type the time used for the annealing step in the PCR reaction.
Elongation Temp	Type the temperature used for the extension step in the PCR reaction.
Elongation Time	Type the time used for the extension step in the PCR reaction.
Melting Temp	Type the temperature used for the denaturation step in the PCR reaction.
Cycles	Type the number of cycles used in the PCR reaction.
MgCl2	Type the concentration of MgCl2 used in the PCR reaction.
dNTP	Type the concentration of deoxynucleotide triphosphates used in the PCR reaction.
Labelled With	Use the drop-down menu to enter the label used for the reaction product, if applicable.
Label Coverage	Use the drop-down menu to specify where the label was incorporated into the reaction product.
Notes	Type any additional information you want stored with this record.

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Northern Samples (Northern Samples)

Assays used in:	Northerns

Sample ID	Type a unique identifier for the sample.
Strain	Type the sample strain. If the sample is from mouse, see Inbred Strains of Mice and Rats for a listing of approved strain names.
Mutations	Type the allele description or symbol for the mutated gene. For tips on determining the official allele symbol in mouse, see Determining standard nomenclature for mouse genes and alleles.
Sex	Use the drop-down menu to specify the gender of the sample, if known, or to indicate pooled samples.
Theiler Stage	If the sample is from mouse, you can use the staging criteria of Theiler to define its age. If you do not know the Theiler stage, fill in the next two boxes. (You can leave them blank if you enter the Theiler stage.)
Age Unit	Use the drop-down menu to specify the age unit.
Age Value	Type the number that specifies the age along with the Age Unit. Leave this field blank if you specify either postnatal or adult as the Age Unit.
Tissue	Type the tissue or structure used for the sample. For mouse, use the Anatomical Dictionary as a reference for appropriate structure names.
Amount Value	Type the number that specifies the amount of the RNA used.
Amount Units	Use the drop-down menu to specify the unit used to measure the RNA amount.
Sample Type	Use the drop-down menu to specify the type of RNA used.
Notes	Type any additional information you want to store with this record.

Note:  If you are planning on submitting data to GXD, we require that you specify the sample age (using either Theiler stages or age units).

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Western Samples (Western Samples)

Assays used in:	Westerns

Sample ID	Type a unique identifier for the sample.
Strain	Type the sample strain. If the sample is from mouse, see Inbred Strains of Mice and Rats for a listing of approved strain names.
Mutations	Type the allele description or symbol for the mutated gene. For tips on determining the official allele symbol in mouse, see Determining standard nomenclature for mouse genes and alleles.
Sex	Use the drop-down menu to specify the gender of the sample, if known, or to indicate pooled samples.
Theiler Stage	If the sample is from mouse, you can use the staging criteria of Theiler to define its age. If you do not know the Theiler stage, fill in the next two boxes. (You can leave them blank if you enter the Theiler stage.)
Age Unit	Use the drop-down menu to specify the age unit.
Age Value	Type the number that specifies the age along with the Age Unit. Leave this field blank if you specify either postnatal or adult as the Age Unit.
Tissue	Type the tissue or structure used for the sample. For mouse, use the Anatomical Dictionary as a reference for appropriate structure names.
Notes	Type any additional information you want stored with this record.

Note:  If you are planning on submitting data to GXD, we require that you specify the sample age (using either Theiler stages or age units).

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RT-PCR Samples (PCR Samples)

Assays used in:	RT-PCR

Note: You must enter the Genes, Primer Pairs, cDNA Generation Methods, and PCR Conditions into the respective worksheets before entering a PCR sample.

Sample ID	Type a unique identifier for the sample.
Strain	Type the sample strain. If the sample is from mouse, see Inbred Strains of Mice and Rats for a listing of approved strain names.
Mutations	Type the allele description or symbol for the mutated gene. For tips on determining the official allele symbol in mouse, see Determining standard nomenclature for mouse genes and alleles.
Sex	Use the drop-down menu to specify the gender of the sample, if known, or to indicate pooled samples that include both sexes.
Theiler Stage	If the sample is from mouse, you can use the staging criteria of Theiler to define its age. If you do not know the Theiler stage, fill in the next two boxes. (You can leave them blank if you enter the Theiler stage).
Age Unit	Use the drop-down menu to specify the age unit.
Age Value	Type the number that specifies the age along with the Age Unit. Leave this field blank if you specify either postnatal or adult as the Age Unit.
Tissue	Type the tissue or structure used for the sample. For mouse, use the Anatomical Dictionary as a reference for appropriate structure names.
Gene	Use the drop-down menu to specify the name of the gene amplified in the PCR reaction.
Primer Pair	Use the drop-down menu to specify the primers used in the PCR reaction.
cDNA Generation Meth	Use the drop-down menu to specify the method used to generate the cDNA used in the PCR reaction.
PCR Conditions	Use the drop-down menu to specify the protocol used in the PCR reaction.
Notes	Type any additional information you want stored with this record.

Note:  If you are planning on submitting data to GXD, we require that you specify the sample age (using either Theiler stages or age units).

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Creating new worksheet fields

To create a new field:

• Position the cursor over the next unused column header in a worksheet.
• Type in the desired name.
• Do not insert a column between previously existing columns since this causes GEN to malfunction!!

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Using assay sheets

Use the assay sheets to:

• Detail the parameters for an experiment or a set of experiments.
• Store the results and images for an experiment or set of experiments.
• Enter data into worksheets using the Manage windows.

Use the blue headings on each assay sheet to link to the Help sheet.

Note: We recommend that you save frequently when using the GEN to prevent data loss in case your computer locks up.

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Creating a new assay sheet

To create a new assay sheet:

1. Click the arrow to the right of Assay Type on the Main Menu to see the drop-down menu. If the drop-down list arrow is not visible, click the Assay Type field to activate it. The drop-down menu lists following assay types:
   • RNA In Situ
   • Immunohistochemistry
   • Northern
   • Western
   • RT-PCR
2. Highlight an assay type and click Create New Assay Sheet. This adds a blank assay sheet of that type to the workbook and opens the new assay sheet.

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Opening assay sheets

To open an assay sheet, click the tab of the desired assay sheet.

Note:  Sometimes the assay sheet opens at the bottom. Use the scroll bar to access the top.

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Adding experimental details to assay sheets

• All fields above Assay Notes contain information on experimental reagents and conditions. These details are stored on worksheets. Each assay sheet uses different worksheets, depending on the data to be collected. See GEN assay sheets and worksheets  for a complete list.
• Enter the details onto the worksheets using either the Manage windows on the assay sheet or by typing them directly in the worksheet fields as described above.
• Once you have entered data on a worksheet, select the appropriate IDs to include pertinent details in the assay sheet. To do this:
  1. Click in a white field such as Gene Name.
     A drop-down menu listing the worksheet items, e.g., genes, appears.
  2. Scroll down the list to choose the item, e.g., the gene, appropriate for that assay.
  3. Tab out of the box. The rest of the fields in this section fill automatically. Any data typed into shaded fields is not recorded in the corresponding worksheet.
  4. Alternatively, you can type the ID for the item in the box. If you type the name of a record that does not exist, a prompt to create one appears and the Manage window opens.
  
  Note:  To manipulate the view of the experimental details on the assay sheet, use the following buttons:

  Item	Description
  > button	Click to expand or collapse the view of experimental details in a section. The expanded view displays all the worksheet entry fields. The collapsed view displays only the ID.
  <  button	Present only for Antibodies. Click to see a collapsed view (only the Antibody Name field appears), an intermediate view (Antibody details appear), or an expanded view (includes both Antibody and Antigen details). The default view shows the antibody, but not antigen, details.

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Using a Manage window to enter experimental details

A Manage window lets you enter and edit worksheet data without leaving an assay sheet. There is no difference in data entered using a Manage window and data entered directly from a worksheet.

To open a Manage window, click the Manage button in each section of an assay sheet (for example, on the RNA In Situ worksheet, click Manage Genes). A list of all fields on the worksheet appears on the right. In the screen shot of the Manage Genes window below, those fields are Name, Symbol, and Notes.

After typing the desired data in the boxes (or selecting from drop-down menus), click Save Changes.

Note: Be sure to save changes before changing window views. If you change views without saving, your edits will be lost.

To create and edit records, use the following buttons on the window.

|<	Display the first record listed on a worksheet.
<	Display the previous record.
>	Display the next record.
>|	Display the last record.
Cancel	Close the Manage window and do not preserve any edits since the last save.
New	Open a new record with all blank fields. By default, the New function is active when you open a Manage window.
Copy	Create a new record with the same data as the current record except Name is blank.
Delete	Remove a record from the worksheet.
Go To	A drop-down menu listing all records in the worksheet. Highlight a name to see that record in the Manage window.
Save Changes	Save the current window data in the corresponding worksheet record. If this is a new record, Save Changes creates a new line for the data in the worksheet. If this is an existing record, changes are saved to the worksheet.

Note: Please keep in mind that the checks on data entry found on the worksheets are not present in the Manage windows.

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Adding specimens/samples

• Specimens are used for in situ and immunohistochemistry assays. Specimens for both assay types are stored on the same worksheet.
• Samples are used for Northern blots, Western blots, and RT-PCR. Samples for each of these assay types are stored on separate worksheets.
• By default, an assay sheet contains a field for one specimen or sample. To manipulate the number of specimen/sample fields, use the following buttons:

Button	Use to ....
Add Specimen	Add a column in the specimen and specimen prep method section.
Remove Specimen	Remove a column in the specimen and specimen prep method sections. To remove the column, highlight any cell in either the specimen or specimen prep method section and click Remove Specimen. If you highlight a cell that is not in the specimen or specimen prep method, an error message appears. Clicking Remove Specimen only deletes a specimen from an assay sheet. The worksheet record is not altered.
Add Sample	Add a column in the sample section and results section.
Remove Sample	Remove a column in the sample section. To remove the column, highlight any cell in the sample section, and click Remove Sample. If you highlight a cell that is not in the section, an error message appears. Clicking Remove Sample only deletes a sample from an assay sheet. The worksheet record is not altered.

• To add specimen/sample details to an assay, type the ID of the desired specimen/sample or select it from the drop-down menu of worksheet entries of that type, and tab out of the field. The other specimen/sample details stored in the worksheet populate the fields automatically.
• If you type in an ID and no record of that name exists, a prompt to create one appears, and the Manage window opens.

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Storing additional experimental details

Use the Assay Notes field to store additional details pertaining to the assay. Text in this field populates the assay sheet directly.

Storing results

The results area is for storing the results of expression experiments—recording the structures stained, expression level, pattern of staining, etc. You can also store images of these results, with accompanying legends, in this area.

For optimal results, we recommend organizing fields in the results section in the following order: images, figure labels and legends, and results. However, not all of these fields are required. You can record results as free text in figure legends or in more detail in tabular format using the <Insert Headings> and <Insert Rows/Bands> functions from the Hide/Show Results menu.

The Hide/Show Results menu button opens or closes the Results menu. Use the Results menu to add the fields to the results section.

The menu has four buttons:

Button	To use...
<Insert Picture>	Adds an image at the highlighted cell. To use: Highlight the cell where you want the upper left corner of the image to appear. This cell must be in the results area. Click <Insert Picture>. A pop-up menu appears. Select the desired image from the directory selection menu. Notes: Be careful where you place the image. Images are not embedded in the worksheet, so they will overlay any text in the cells underneath. However, if this happens, you can move an image by clicking on it and dragging it to a new position. You can resize images by clicking on a corner and dragging. Most image formats are compatible with the GEN, but some work better than others. Adobe files are not compatible with GEN. Excel converts any images inserted into the notebook into a PICT file. TIFF images take approximate ten times more memory than JPGs.
<Insert Legend and Label>	Adds a single row for a figure label and a box directly beneath for the legend. To use: Highlight a cell in the row where you want to add the label. Click <Insert Legend and Label>. Type the desired label and legend in the boxes provided. Note:   If you are planning on submitting data to GXD, it is helpful for the figure legend to include details necessary for interpreting the image, such as plane of section, area pictured, etc.
<Insert Headings>	Adds headings to the results section to organize your expression results. To use: Highlight a cell in the row where you want to add the headings. Click <Insert Headings>.
<Insert Rows> or <Insert Bands>	<Insert Rows> appears in the results menu for RNA in-situ and Immunohistochemistry assays. It adds the number of rows you designate. These rows are for recording your expression data and contain all the drop-down menus appropriate for the headings. <Insert Bands> appears in all blot assays. It adds rows for describing the bands on a blot. To use: Highlight the row directly below the headings Click <Insert Rows> or <Insert Bands>.

Note:

• It is important to highlight a cell in the row where you wish to insert the label and legend, headings, or rows/bands. If you insert a field into another field, the second field will be displaced or divided and you may cause a run-time error.
• If you insert any of the results fields in the wrong space, you can delete them.
• To delete a results field:
  1. Highlight all the white columns of all the rows you wish to delete. Excel does not allow partial deletion of a row.
  2. Click Edit—>Delete. Using the Delete button does not remove the field.
  3. Choose Shift cells up from the dialog box and click OK.

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Recording results for in situ and immunohistochemistry assays

Headings and rows are used in the results section to organize and record expression results. There are six headings. Each row contains the drop-down menus appropriate to each heading. Enter expression results in the rows. Use as many rows as needed to describe each specimen. If you want to add additional results headings, see Customizing GEN to your needs.

Results heading	To use...
Specimen	Select the appropriate specimen from the drop-down menu. The drop-down menu includes only specimens added to the Specimens Used section of the assay sheet.
Structure	Type the anatomical structure analyzed. For mouse, you can use the Anatomical Dictionary as a reference for appropriate structure names. It lists the tissues and structures for each developmental stage (Theiler stage) in a hierarchical fashion. You can cut and paste the complete name and the ID for the anatomical structure, e.g., "TS15;1st arch;mandibular component [1011]," into the Structure cell. This enables standardization of structure nomenclature between researchers and facilitates entry of results into GXD. If the dictionary does not fit your needs, you can use your own anatomical terms.
Expression Level	Select the appropriate adjective from the drop-down menu to describe the level of expression in the structure analyzed.
Pattern	Select the appropriate adjective from the drop-down menu to describe the pattern of staining in the structure analyzed. Regionally restricted is useful when staining is restricted to part of the structure analyzed, and Not applicable (or blank) is appropriate when the staining is Absent.
Image Label	Indicate which image pane illustrates the result, if applicable.
Note	Type additional remarks to further describe the staining.

Recording results for blot assays

The Results section has three headings for organizing results. Each row contains drop-down menus appropriate to each heading. Use the rows to describe the bands seen on the blot.

Results heading	To use...
Band Size	Type in the size of the band.
Band Units	Select the appropriate unit of measurement from the drop-down menu.
Sample ID	Select the appropriate adjective from the drop-down menu to describe the level of expression of that band seen in the sample.

Note:   To indicate control bands, you can either note this in the Results Notes field or you can type control in the Sample ID field.

For blot assays, you can also use the Results Notes field to describe your expression results.

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Copying information into a new notebook

You may find that additional workbooks are necessary if:

• there are many images or assay sheets in a single workbook and memory limitations are an issue
• you want separate workbooks for different projects
• you need a workbook for a new lab member.

When you create an additional workbook, it contains all the data previously entered in the worksheets of an existing workbook. This allows you to avoid reentering methods, reagents, or specimens.

To generate a new workbook:

• From the Main menu, click Copy info to new Workbook.
• Type the name of the new workbook and the place you want to store it in the dialog box.

The new workbook contains all the information in the parent workbook except the assay sheets.

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Determining standard nomenclature for mouse genes and alleles

We encourage you to use the official gene, allele, and mutation symbols and names as approved by the International Committee on Standardized Genetic Nomenclature for Mice.

A simple method to find the approved gene symbol and name is to use the quick gene search at Mouse Genome Informatics. You can also search for a gene symbol with a sequence accession number. If you cannot find the gene, include in the Gene worksheet Notes field any information that will be useful to help us identify the gene and assign a new symbol and name if necessary. Types of useful information include: references, gene structure or function, gene family information, homologies to other species, etc.

To find the approved allele symbol and name, you can use the Phenotypes, Alleles, and Disease Models Query Form at Mouse Genome Informatics. (For a quick guide to the nomenclature, see the Allele and Mutant Quick Nomenclature Guide. If you cannot find the allele, include in the Specimen or Sample worksheet notes field any information that will be useful to help us identify the allele and assign a new name if necessary. Types of useful information include: references, type of mutation, lab generated in, etc. Note: At this time GXD does include information from knock-ins and knock-outs but not from other kinds of transgenic animals.

In GXD we use the Anatomical Dictionary to describe the structures expression was analyzed in. We encourage you to do the same.

Finally, to submit data to GXD, you must make entries in certain fields. The validation function insures these fields are filled in. See Validation in the GEN for further details.

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Validation in the GEN

The validation feature is designed to help you check data you wish to submit to GXD. It looks at fields that we think are key to having a complete entry to make sure there is data in them. For instance, the validation feature looks to ensure that each specimen has either an age or Theiler stage specified. Anything that we believe is an omission will be listed on the Validation Report. You do not have to validate data in order to use GEN or even to submit data to GXD.

You can validate a worksheet, an assay sheet, or the entire workbook.

Location	Purpose	To use...
Validate [worksheet name] button at the top of some worksheets	Validate the worksheet.	This function is available on worksheets that have fields to validate (Immunohistochemistry and In-Situ Specimens; Immunohistochemistry and In-Situ Specimen Prep; Northern Samples; Western Samples; and PCR Samples). Click Validate [worksheet name]. If there are no omissions on this worksheet, nothing will happen. If there are omissions, GEN goes to the Validation Report sheet. This page lists the worksheet or assay sheet and describes the omission. Return to the worksheet you are trying to validate by clicking on its tab. A message asks, "Do you want to clear the Validation Report?" If you choose Yes, GEN removes the text describing the validation errors from the Validation Report. If you choose No, the listed omission remains on the Validation Report until you clear the page, whether or not you correct the omission. Correct the omission on the worksheet.
Validate Submission button at the top of every assay sheet	Validate the assay sheet.	Click Validate Submission. If there are no omissions of crucial data associated with this assay, nothing happens. If there are omissions, GEN goes to the Validation Report sheet. This page lists the worksheet or assay sheet and describes the omission. Click the tab of a worksheet or an assay sheet with an omission. A message asks, "Do you want to clear the Validation Report?" If you choose Yes, GEN removes the text describing the validation errors from the Validation Report. If you choose No, the listed omission remains on the Validation Report until you clear the page, whether or not you correct the omission. Correct the omission on the worksheet or assay sheet. If there was more than one omission and you did not clear the text from the Validation Report, you can return to the Validation Report to review the next omission to correct. If you do not clear the Validation Report, each time you leave that page to go to another page in the workbook, a message asks, "Do you want to clear the Validation Report?"
Validate this Workbook on the Main menu	Validates every assay and worksheet in the notebook.	Click Validate this workbook. If there are no omissions of crucial data in the workbook, a message informs you of that. If you click OK, the Main menu reappears. If there are omissions, the box lists the number of errors found in the workbook. Click OK to go the Validation Report sheet. This page lists the worksheet or assay sheet and describes the omission. Click the tab of a worksheet or assay sheet with an omission. A message asks, "Do you want to clear the Validation Report?" If you choose Yes, GEN removes the text describing the validation errors from the Validation Report. If you choose No, the listed omission remains on the Validation Report until you clear the page, whether or not you correct the omission. Correct the omission on the worksheet or assay sheet. If there was more than one omission and you did not clear the text from the Validation Report, you can return to the Validation Report to review the next omission to correct. If you do not clear the Validation Report, each time you leave that page to go to another page in the workbook, a message asks, "Do you want to clear the Validation Report?"

Note:  If you perform validations in a notebook with an uncleared Validation Report, any new errors appear on the list after the old errors.

Certain assumptions were made when designing the validation feature that may not be correct in all instances. For instance, it was assumed that each in situ and immunohistochemistry assay would contain an image. Therefore, if you enter one of these assays and do not enter an image, you will get an error message telling you that you have failed to enter an image label. You can safely ignore this, and any other error messages that do not apply to your entry.

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Submitting data to GXD

The GEN includes a mechanism for voluntary submission of data to the Gene Expression Database (GXD). Electronic submission both increases the exposure of your work and the amount of data available to the scientific community. In addition, it strengthens GXD: the value of GXD is directly proportional to the amount of data it contains. Submitted data receives accession numbers that you can cite in publications and grant applications. In this manner, data that is not complete enough to warrant a standalone publication or that cannot be included in a publication due to space limitations can be submitted to GXD, accessed and analyzed by other researchers, and referenced.

Note:  Submission is entirely voluntary; the data and time of submission are at your discretion. You can choose to submit an individual assay or group of assays while retaining others to submit later (or not at all).

To create a submission notebook:

1. For ease, we recommend that you validate the assays you propose to submit before creating a submission file (although you can validate the submission notebook).
2. Click Create New Submission on the Main menu. A list of every unsubmitted assay in the workbook appears.
3. Click the check box beside each assay you want to submit at this time.
4. Click Create Submission. A dialog box appears.
5. Enter a name for the submission file and the location to store it in.
6. Click Save to create a submission notebook. The submission notebook closes and the notebook you created the submission from remains open.

There are two ways to open the submission notebook:

1. Double-click the submission notebook icon.
2. From the Excel File menu, select Open (click File—>Open), then select the submission file.

The submission notebook is exactly like a normal workbook except:

• There is no Main menu. You can add additional probes, specimens, results, etc., to this submission and you can validate individual assay sheets, but you cannot create new assay sheets or perform any other functions of the Main menu.
• The only data it contains are from assays selected for submission. This means, for example, that if a probe is not used in the selected assays, GEN removes it from the Probes worksheet.

Be sure to delete any data from Notes fields that you do not want made public, since any notes you've entered in assay sheets or worksheets will appear in the created submission.

To submit your data:

• Please send your validated submission notebook to GEN@informatics.jax.org as an e-mail attachment.
• After we receive it, we will confirm receipt of your submission.
• Once we review your data and enter it into GXD, we will e-mail you the accession number of your submission and detail where you can view your data.

You can also send any questions regarding submissions to the same address: GEN@informatics.jax.org.

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Customizing GEN to your needs

You can customize the Gene Expression Notebook to meet your specific needs. Instructions for various modifications appear below.

Note:  If you use Excel functionality to modify the Notebook in ways other than those listed below, you may encounter unexpected results.

Can I adjust column widths?	Some worksheets have more columns than you can view on a single "page." You can modify the column width so you can see more columns or make some not visible at all. There are two ways to do this. To change a column width by dragging: Click the column heading to select the entire column. Move the mouse to the right edge of the box until you see the cross-hairs (arrows pointing left and right). Hold the mouse button down and drag the column to widen or narrow the view. To change a column width using Excel menus: Select a field in the column to be adjusted. Click Format—>Column—>Width. Type the desired width in the dialog box and click OK.
How do I add additional fields to worksheets?	To do this, type a new field heading in the row after the last field heading. The heading is saved and copied to new Notebooks or Submissions. Avoid inserting new columns within the provided columns since this causes the GEN to malfunction. The new field headings do not appear on the assay sheet or the Manage window.
How do I add additional headings to the results section?	To do this, type a new heading in the shaded row after the last heading. Avoid inserting new columns within the provided columns since this causes GEN to malfunction. The new results headings appear only on the modified assay sheet. Any new assay sheets do not contain the additional header. Only add additional results headings for In Situ and Immunohistochemistry assays. Additional headings added to blot assays are overwritten if you add more samples to the assay.
How do I record the control lanes in my blots?	You can create samples for your molecular weight standards, plasmids, etc. It is likely that these "samples" will not validate properly, but you can ignore that. Alternatively, you can make a notation of non-result blot lanes in either the results or assay notes field.
How do I modify the names of the navigational tabs?	You can only partially change the name of the assay sheet navigational tabs. You should not change the name of the worksheet navigational tabs. The assay sheet tabs list the type of assay followed by a number in parentheses. You can delete the number designation and/or add additional text, but if you alter the assay type designation, the notebook will malfunction. To rename: Double-click on the tab name to highlight it. Press the right mouse button (control-click for a single button mouse) to deselect and place the cursor after the current name. Delete the number or add additional text. [For example, you could change RNA In Situ (2) to RNA In Situ E13.5.]
How do I delete worksheet or results entries?	You can delete entries in an assay results section or a worksheet row. Note:   Never delete columns in the Gene Expression Notebook. To delete a worksheet row or a portion of a results table: Highlight the entire active portion of the row (any column with a worksheet header or any white field in a results table). If you do not highlight the entire active row, heading, label, or legend, you cannot delete it. Click Edit—>Delete. Select Shift rows up when the dialog asks how to deal with the deleted cell. Note:  You cannot delete using the Delete key on your keyboard. You must use the Excel menu.
How do I delete assay sheets?	Go to the assay sheet you wish to delete. Click Edit—>Delete Sheet. Note:   You cannot undo this function once performed.
I have my data in a previous version of GEN. How can I get it into the newest version of GEN?	Unfortunately, you cannot simply import data into another version of GEN; you will need to cut and paste your data into the new GEN. Tip: If you do the worksheets first, the specimen data will automatically fill in when you copy the assay sheets.

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Troubleshooting the GEN

Problem	Probable Cause	Suggested Remedy
I saved my data and closed my notebook but now I can't find it.	You saved the GEN to an unexpected location. This can happen if you select Open as File instead of Save to Disk when downloading the GEN initially.	Use your computer's Search function to find the GEN.xls file.
GEN will not open, locks up, shuts down unexpectedly, or gives you a run-time error message.	For all users: your Excel application needs an update. For OS9 users: the Excel application on your Mac may not have enough memory assigned.	Verify that you have installed the latest updates for your version of Excel. To make sure that your computer has all the necessary updates go to http://office.microsoft.com/officeupdate/default.aspx and click on "Check for Updates. In OS9, increase the memory as follows: Close the Excel program. Highlight the Excel file. (Highlight only; do not activate the program.) From the menu at the top, click File—>Get Info. A dialog box appears. Locate the Memory Requirements section and type the amount of memory desired in the Minimum Size and Preferred Size fields, making sure that Preferred is larger than Minimum. Close the window. Excel now has the reassigned memory.
I've installed Excel updates/increased the amount of memory, and I'm still having problems.	The size of the file has exceeded the capacity of Excel.	If you have many images in multiple assays, you may still exceed the ability of the program. In this case, create a new notebook. See Copying information into a new workbook.
I can't get an image to insert in the GEN.	Either the image file is incompatible with Excel or there is a memory problem.	Most image formats are compatible with the GEN, but some work better than others. Adobe Photoshop files do not work with the GEN. Try saving the image as a PICT file and reinserting it. If the problem persists, increase the memory or create a new workbook.
My worksheet entry doesn't appear on the drop-down menu on the assay sheet.	The record is either lacking an ID or you skipped a row on the worksheet.	If a record lacks an ID, it will not appear on the drop-down menu. Sometimes if the worksheet page does not open all the way to the left, you may forget to enter a name in the first field. Alternatively, if you leave a blank row between entries on a worksheet, the records below the skipped row will not appear on the drop-down menu. In very rare instances, your entry will fail to appear because the GEN suffered a momentary glitch when generating the drop-down menu. In those cases, you can type the entry id (as it appears on the worksheet) into the assay field; the other fields will still fill in when you tab out.
When using the Manage windows on the assay sheets to create an entry, the cursor appears as an hourglass.	This happens in some versions of Excel on the Mac and is harmless.	The cursor is still active even though it may appear as an hourglass when you are using the assay sheet Manage windows.
I'm trying to insert Headings or Legends and Labels into the results section of my assay, but I'm getting a run-time error message.	Excel thinks you want to merge incompatible files.	If you do not select the cell in the results area where you want the Headings or Legends and Labels to be inserted, Excel may try to insert the heading/legend in the cell you last selected. Since these items have different sizes and numbers, trying to insert a Heading into a Legend and Label (or vice versa) causes an error. To prevent the problem, select the cell in the results where you want the Headings or Legends and Labels to be inserted.
I would like a paper copy of an assay record in my GEN. When I ask the assay to print, I get massive numbers of pages with gray boxes on them.		To print an assay sheet, you need to set the print area. Highlight the area of the assay sheet you wish to print. Click File—>Print Area—>Set Print Area to choose this area for printing. Click Print.
I've double-labelled a specimen, but I can't figure out how to get both genes and probes into one assay sheet.		The GEN is designed so that an assay describes the results obtained using one probe or antibody. (The exception is the RT-PCR assay, which allows you to enter the results from more than one primer pair.) To record the results from a double (or triple) labeling experiment, you'll need to fill out a separate assay sheet for each probe or antibody used.
The TIFF image I've inserted in my assay is incredibly large.		You can resize a TIFF image by clicking on its corner and dragging.
Some of my worksheet columns have disappeared.	When resizing a column, you dragged it so that it covers other columns.	To display hidden columns: Select cells in the columns to the left and right of the hidden columns. Click Format—>Column—>Unhide. Repeat as necessary for any additional hidden columns. Note: If your rows should disappear, follow a similar procedure, selecting the rows above and below the hidden ones and clicking Format—>Row—>Unhide. Alternatively: Select cells in the columns to the left and right of the hidden columns. When the cursor changes to a vertical line with cross hairs (left and right arrows), move it slightly to the left or right until it changes to a double vertical line with cross hairs. Click and hold the mouse button, then drag the cursor to the right. The covered column appears. Repeat for all covered columns. (This becomes easier with practice.) If the first column (or row) of a worksheet is hidden: Click Edit—>Go To. Type A1 in the Reference box. Click OK. Click Format—>Column—>Unhide (or Format—>Row—>Unhide).
When I open the Create New Submission dialog box, not all the assays present in my workbook appear.	The GEN is designed to prevent you from submitting the same assay twice.	Once an assay has been included in a submission, it is no longer included in the list of assays on the submission creation form. If you want it to reappear on the list, go to the assay sheet and delete the word Submitted in the column beside the Validate Submission button.
How can I suggest improvements for future versions of the GEN?		You can contact us at GEN@informatics.jax.org. We welcome all comments (positive or negative) from users.

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Getting help

This documentation is available on the Web at http://www.informatics.jax.org/mgihome/GXD/GEN/gen.shtml. You can get additional help by contacting GEN@informatics.jax.org.

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Getting started using the GEN (tutorial)

To help you get started using the GEN, the following example shows how to enter data from an RNA in situ assay from start to finish. For this exercise, let's assume that you have performed nonradioactive RNA in situs on E10.5 tissue from wild-type and Shh mutant mice using a probe for the Bmp4 gene. In the wild-type embryo, you have staining in the limb—in the margins of the limb and in the apical ectodermal ridge—and at high levels in the somites. In the mutant embryo, the staining is still present in the apical ectodermal ridge but not in the margins of the limb or in the somites.

1. Create an assay sheet for your experiment.
• Go to the Main menu. If you just opened the GEN, the Main menu appears. Otherwise, to access the Main menu, click the Menu tab at the bottom of the page.
• Click in the white box to the left of Create New Assay Sheet. A drop-down list arrow appears next to the white box.
• Click the drop-down list arrow. A menu of the assay types available in GEN appears.
• For a PC, click RNA In Situ.
• For a Mac, click the drop-down list arrow, hold down the (left) mouse button, and drag to RNA In Situ.
• Click Create New Assay Sheet to create and open an assay sheet to store your experiment in. The assay sheet is entitled RNA In Situ (#). If this is the first in situ assay stored in the GEN, it will be RNA In Situ (2).

Experimental details are used in assays and stored in worksheets. There are two methods for entering worksheet data: using the Manage windows or directly into a worksheet. Steps 2-4 are examples of data entry using Manage windows; steps 5 and 7 demonstrate entering data directly into a worksheet.


2. Type the name of the gene whose expression you analyzed in your experiment into the assay sheet.
   • Often the assay sheets open at the bottom of the page. If this happens, scroll to the top of the page to start your entry.
   • Click Manage Genes in the upper-left corner. The Editing: Genes dialog box appears.
   • Type Bone morphogenetic protein 4 in the box labelled Name. Type Bmp4 in the box labelled Symbol. If there is any other information you want to add regarding the gene analyzed, put it in the Notes box.
   • Click Save Changes (otherwise the entry will be lost). The data is entered into the Genes worksheet. (You can view this directly by clicking on the Genes tab at the bottom of the assay sheet page.)
   • Close the window or click Cancel.
   • As you did when creating your assay sheet, click the white box to the right of Gene Name. A drop-down list arrow appears.
   • Click the drop-down list arrow and select Bone morphogenetic protein 4 from the menu of gene names. Tab out of the box. The remaining fields in the Gene section fill in automatically.


3. Type the name of the probe you used in your experiment.
• Click Manage Probes. The Editing: Probes dialog box appears.
• In the first box, type something similar to what you'd use in referring to your own probes, such as Bmp4 Probe 1. Since the entries in this box will appear in the drop-down menus, each probe name must be unique for the GEN to function properly.
• In the Gene box, type Bmp4. This should be the name of the gene the probe was derived from.
• Select cDNA from the Sequence Type drop-down menu. If the option you want is not on the list, type it into the box.
• Type Mouse into the Species box.
• The following boxes further define the probe specificity. Type D14814 into the Sequence Acc # box, NA 5600-5724 into Subsequence, and Third Exon into Region Covered. Put any other information you want to add regarding the probe in Notes; for example, synthesized 3/11/07; not hydrolyzed.
• Click Save Changes, close the dialog box, and select Bmp4 Probe1 from the drop-down menu of probe names on the assay sheet. Tab out of the box. The remaining fields in the Probe section fill in automatically.


4. Next, type the method you used to prepare your probe into the assay sheet.
• Click Manage Probe Preps. The Editing: Probe Prep dialog box appears.
• In the first box, type the name of the method you used to make your probe, such as Nonradioactive. Each probe prep method name in the GEN must be unique.
• Use the drop-down menus to fill in the other boxes. For this exercise, for Type, choose RNA; for Hybridization, choose Antisense; for Labelled With, choose Digoxigenin; and for Visualized with, choose Alkaline phosphatase. For any of these values, if the option you want is not listed, you can type it into the box.
• As above, click Save Changes, close the dialog box, and select Nonradioactive from the drop-down menu of Probe Prep Methods on the assay sheet. Tab out of the box for the remaining fields in the Prep Method section to be filled in automatically.


5. Add your specimens to the GEN using the Specimens worksheet.
• As you become an experienced GEN user, you'll probably start adding your experimental details directly to the various worksheets (rather than using the Manage window) since entry is usually quicker that way and you can review what you already have easily. The following instructions, therefore, illustrate how to enter data using a worksheet.
• Click the Specimens tab at the bottom of the assay sheet to go to the Immunohistochemistry and In-Situ Specimens worksheet.
• To enter your wild-type specimen, type a name in the first field under Specimen ID similar to what you'd use for your tissue samples, such as 2/25/07 E10.5 WT #1. Each in situ/immunohistochemistry specimen name in the GEN must be unique.
• Type C57BL/6J into the Strain field.
• Type WT into the Mutations field (or leave it blank).
• Type 17 into the Theiler Stage field. (Alternatively, if you fill in the age unit and value boxes, feel free to leave this blank.)
• Choose embryonic day from the Age Unit drop-down menu and type 10.5 into the Age Value field.
• Choose 0.5 from the Noon of Plug Day= drop-down menu.
• Choose Unknown from the Sex drop-down menu.
• If there is anything else you want to add regarding the specimen, put it in the Notes field.
• Follow essentially the same procedure to add the mutant specimen in the second row fields. For the Specimen ID, use 2/25/07 E10.5 mut #1. For the Strain, use 129S1/Sv and 129X1/SvJ. For Mutations, use Shh^tm1Chg/Shh^tm1Chg. In the Notes box, write targeted mutation generated by Chin Chiang. For all other boxes, use the values for the wild-type specimen.


6. Add the specimens to your experimental record.
• Click the RNA In Situ(2) tab to go to the assay sheet.
• You need as many white boxes on the Specimen ID line as you have samples in your assay. For this exercise you need two, so click Add Specimen once.
• Click in the first box to make the drop-down list arrow to appear next to the box. Select the WT specimen from the drop-down menu and tab out of the box. Repeat this in the next box to add the mutant specimen to the assay sheet.


7. Add the details of how you prepared the specimens to the experimental record, using the worksheet.
• To open the Immunohistochemistry and In-Situ Specimens worksheet, click the Specimen Preps tab.
• In the first field, type the name of the method you used to prepare your probe, such as Para fixed WM. Each specimen prep method name in the GEN must be unique.
• Use the drop-down menus to fill in the other fields. For this exercise, for Type, choose Whole mount; for Fixation, choose 4% Paraformaldehyde; and for Embedding, choose Not applicable. In the Notes field, you can type para o/n, then 20% sucrose in PBS until sank. For any of the values with a drop-down menu, if the option you want is not listed, type it into the field.
• Click the RNA In Situ tab. Click in the first box following the Spec Prep Method, choose Para fixed WM from the drop-down menu that appears, and tab out of the box. Repeat for the mutant specimen.


8. Add any other details of the experimental procedure that you want recorded in the Assay Notes box.



9. Add your results to the experimental record.
• For optimal results, add the results fields in this order: Image; Figure legend and label; then Results.
• The GEN does not contain an image for this exercise, so if you do not have an image on your computer to use, skip straight to Adding your results.
• In all cases, the first step is to click Hide/Show Results Menu.

Adding an image of your results:
• Click in the results field to select the area where you want the upper left corner of the image.
• Choose <Insert Picture> from the RNA In Situ: Results list box.
• Choose the image file from the selection window. Click Open to select it for insertion. The image is inserted into the results area.
• You can move this image by (left) clicking on it and dragging to a new position.

Adding a label and legend to your image:
• If you wish to add a legend for your image, click in the desired row in the results field below the image.
• Choose <Insert Legend and Label> from the RNA In Situ: Results list box.
• Add a label to the first line and add the legend in the box below that.

Adding your results:
• Click in the results section to select the row where you want to add the headings.
• Choose <Insert Headings> from the RNA In Situ: Results list box. This adds column headers to help you organize your results.
• When you add the headings, one blank row is also added for recording results.
• You can add more rows by choosing <Insert Rows> from the RNA In Situ: Results list box. Generally, you should add one row for each structure analyzed in each specimen. For this exercise, you will need four lines.
• At this point, you should probably close the RNA In Situ: Results list box. You can reopen it at any time by clicking Hide/Show Results Menu.
• Use the drop-down menu to choose the first specimen whose staining pattern you want to describe. For this example, choose 2/25/07 E10.5 WT #1.
• In the structure box, type the name of a structure you analyzed. For this example, use limb. You can use The Mouse Anatomical Dictionary as a reference for appropriate structure names. You can cut and paste the complete name and ID for the anatomical structure, e.g., TS17;limb[2095] or TS17;forelimb bud;ectoderm;apical ectodermal ridge [2098](AER) or TS17;hindlimb bud;ectoderm;apical ectodermal ridge [2102](AER).
• To describe the intensity of the staining, use an adjective from the Expression Level drop-down menu. For this specimen, use Strong.
• To describe the Pattern of the staining, use a descriptor from the Pattern drop-down menu. Since in this specimen, only part of the limb was stained, choose Regionally restricted.
• If there is an image of this embryo on the assay sheet, type the label of that image in the next box.
• In the Note box describe how the staining was regionally restricted, such as strong expression in the posterior and anterior margins of the limb, including in both the ectoderm and the underlying mesenchyme. Also present in the apical ectodermal ridge.
• On the next line of the results field, describe the staining in the somites. For specimen, choose 2/25/07 E10.5 WT #1. For structure, type somites. For Expression Level, choose Strong. For Pattern, choose Homogeneous.
• On the next line of the results field, describe the staining pattern in the mutant embryo. For specimen, choose 2/25/07 E10.5 mut #1. For structure, type limb. For Expression Level, choose Present. For Pattern, choose Regionally restricted. In the Note box, type staining present in the apical ectodermal ridge but absent from the posterior and anterior margins of the limb.
• On the next line of the results field, describe the staining in the mutant somites. For specimen, choose 2/25/07 E10.5 mut #1. For structure, type somites. For Expression Level, choose Absent. Leave the Pattern field blank.

Congratulations! You have now successfully entered an experiment into the GEN.

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