NCI Microarray Manual

##### 

**Introduction**

The use of DNA microarrays for gene expression analysis has become a
powerful tool in the arsenal of the molecular biologist. This workshop
and manual are designed as an introduction to the use of microarrays.
Because of the dynamic nature of this topic, a comprehensive treatment
of microarray technology is not feasible in this format. A broad range
of diverse protocols exists that cover every aspect of microarray
technology, and we encourage you to investigate new techniques on your
own. However, we will endeavor to provide you in this training class
with sound methods that should work successfully in your subsequent
experiments.

In general, an *[array]{.underline}* is defined as an ordered
arrangement of known DNA sequences on a solid substrate. A
*[macroarray]{.underline}* is composed of sample spots with diameters of
300 microns or larger that can be easily imaged by existing gel and blot
scanners. Macroarrays are usually printed on nylon membranes and the
features are spaced far enough apart to allow for radioisotopic
detection. A *[microarray]{.underline}* has features typically less than
200 microns in diameter with thousands of spots and is usually printed
onto a coated glass microscope slide. A microarray also requires
specialized high resolution scanning equipment for data collection.
Array hybridization chemistries are similar to those of standard nucleic
acid hybridization. The techniques used are based on Northern and
Southern blotting, but rather than only a handful of genes being
examined at a time with these traditional methods, thousands of gene
sequences can be detected simultaneously in a microarray.

Two main variants of microarrays are presently in use, the cDNA array
and the oligonucleotide array. Arrayed cDNAs are typically PCR-amplified
fragments from cDNA libraries or clone collections that are robotically
spotted onto glass slides. Oligonucleotides may be anywhere in length
from 25- to 80-mers and are either synthesized *in situ* on silica
substrates or presynthesized and deposited on glass. Glass is the
substrate of choice because it gives a rigid, thermostabile, optically
flat surface for high-density arrays. Many coatings and surface
chemistries have been developed for arraying DNA fragments, but the
common feature of all of them is the presence of chemically active
groups available to bind either the DNA or a linker group on a
synthesized oligonucleotide.

The individual DNA molecules on the array are referred to as
*[probes]{.underline}* because they are the known sequence. The
experimental sample of mRNA that is reverse transcribed into cDNA with
labeled nucleotides is considered the *[target]{.underline}* as it is
uncharacterized. This terminology is often confused because it is the
probe that is immobilized and the target that is in the hybridization
solution, the reversed of traditional blotting techniques. To avoid
confusion, refer to the experimental sample as the target, or the
"labeled cDNA".

Oligonucleotide arrays from Affymetrix are used with a single RNA source
per hybridization. On the other hand, spotted cDNA and spotted oligo
arrays are hybridized with two RNA sources that are each labeled with a
different fluorescent dye. The competitive hybridization between these
two RNAs is analyzed by comparing the ratio of the intensity of the two
fluors (such as Cy3 and Cy5). Because a ratio is used, experimental
results can be compared across multiple arrays despite slight variations
in the DNA probe concentration on the array from different print sets.
Many RNA labeling protocols are currently employed for use with
microarrays. The RNA can be labeled using reverse transcriptase to
directly incorporate nucleotides covalently linked to fluorescent
molecules. While this method is a little simpler, the bulky fluors do
not always incorporate efficiently during the transcription, often
resulting in biased incorporation of the Cy3- over the Cy5-labeled
nucleotide. In an effort to overcome this, the cDNA can be indirectly
labeled by enzymatic incorporation of amino allyl-modified and/or amino
hexyl-modified nucleotides into the cDNA followed by chemical coupling
of the Cy3 and Cy5 fluors to the amino allyl/hexyl groups. This labeling
is more efficient and less biased than direct incorporation labeling as
the amino allyl/hexyl groups are smaller and less bulky than the
fluorescent nucleotide molecules.

It cannot be emphasized enough that the quality of your RNA will
determine the success of your microarray. While hundreds of variables
exist that will affect microarray results, the most common problem
continues to be poor quality or degraded RNA in the initial labeling
reaction. Additionally, too small a quantity of RNA in the labeling is
often a contributing factor to microarray experimental failure. It is
outside the scope of this workshop to teach RNA isolation techniques,
but any method that gives intact RNA with a 260/280 ratio \>1.8 should
be sufficient. If it is not possible to get an adequate amount of RNA
(\>10ug), published protocols for RNA amplification and signal
amplification techniques are available. We do suggest that you master
the techniques presented in this workshop before you try more
technically challenging methods.

+-----------------------------+-------------------+-------------------+
| Recommended Supplies for    |                   |                   |
| Microarray Labeling and     |                   |                   |
| Hybridization               |                   |                   |
|                             |                   |                   |
| (*March 2005)*              |                   |                   |
+-----------------------------+-------------------+-------------------+
| **SUPPLIES**                | **SUPPLIER**      | **Catalog No.**   |
+-----------------------------+-------------------+-------------------+
|                             |                   |                   |
+-----------------------------+-------------------+-------------------+
| SuperScript™ Indirect cDNA  | Invitrogen        | L1014-02          |
| Labeling Kit                |                   |                   |
+-----------------------------+-------------------+-------------------+
| Dyes:                       | Pharmacia         | PA23001           |
|                             |                   |                   |
| Cy3 monofunctional reactive | Pharmacia         | PA25001           |
| dye                         |                   |                   |
|                             |                   |                   |
| Cy5 monofunctional reactive |                   |                   |
| dye                         |                   |                   |
+-----------------------------+-------------------+-------------------+
| Ribonuclease H (RNase H)    | Invitrogen        | 18021-071         |
+-----------------------------+-------------------+-------------------+
| Formamide,                  | Any reputable     | We recommend      |
|                             | chemical          | buying small      |
| molecular biology grade,    | manufacturer      | aliquots, ≤ 100ml |
|                             |                   | bottles           |
| (deionized if available)    |                   |                   |
+-----------------------------+-------------------+-------------------+
| Coverslips for 48-pin       | Erie Scientific   | 25X60I-2-4789     |
| print:                      |                   |                   |
|                             | Erie Scientific   | 25X60I-M-5439     |
| Lifterslips™ (25 X 60 mm)   |                   |                   |
|                             |                   |                   |
| or mSeries™ (25 X 60 mm)    |                   |                   |
+-----------------------------+-------------------+-------------------+
| Staining Dish/rack (10      | Fisher            | 08-812            |
| slide)                      |                   |                   |
+-----------------------------+-------------------+-------------------+
| Slide Box (100 slide)       | Thomas Scientific | 6708-G28          |
+-----------------------------+-------------------+-------------------+
| Slide Box (25 slide)        | Thomas Scientific | 6708-G08          |
+-----------------------------+-------------------+-------------------+
| Hybridization chambers:     | Genomic Solutions | JHYB200004        |
|                             |                   |                   |
| > Dual Hyb Chamber          | Telechem Int.,    | AHC               |
| >                           | Inc.              |                   |
| > Single Hyb Chamber        |                   | 2551              |
| >                           | Corning           |                   |
| > Single Hyb Chamber        |                   | AHCXD             |
|                             | Telechem Int.,    |                   |
| Deeper hyb chamber to       | Inc.              |                   |
| accommodate thicker         |                   |                   |
| mSeries™ cover slip:        |                   |                   |
|                             |                   |                   |
| > Single Hyb Chamber        |                   |                   |
+-----------------------------+-------------------+-------------------+
| Hyb Oven                    | Fisher Scientific | 13-247-10         |
+-----------------------------+-------------------+-------------------+
| Forceps                     | Fisher Scientific | 10-295            |
+-----------------------------+-------------------+-------------------+
| Mini-Elute PCR Purification | Qiagen            | 28004             |
| Kit                         |                   |                   |
+-----------------------------+-------------------+-------------------+
| High Quality Pre-filtered   | Invitrogen        | 15561-020         |
| BSA                         |                   |                   |
+-----------------------------+-------------------+-------------------+
| Centrifuge with microplate  |                   |                   |
| carrier assembly            |                   |                   |
+-----------------------------+-------------------+-------------------+

**[Target Preparation/Hybridization Using Total RNA]{.underline}**

 

***I. [cDNA Generation:]{.underline}***

Prepare separate cDNA labeling reaction for each fluorescent dye you
wish to use.

A "master mix" (step 4) can be made, and the reaction increased up to 5X
if needed.

1.  For each dye dilute total RNA to between 5.0 -- 20.0 µg in 16.0µl of
    DEPC water (0.3- 1.25 µg/µl). 

```{=html}
<!-- -->
```
1.  Add 2.0µl of 2.5μg/µl anchored oligo d(T)~20~ primer.

2.  Incubate at 70°C for 5 minutes. Cool on ice for at least 1 min.

3.  Combine the following components for each sample in a sterile,
    RNase/Dnase-free microcentrifuge tube:

> a.        6.0µl of 5X First-Strand buffer
>
> b.       1.5µl of 0.1 M DTT
>
> c.        1.5µl of 10mM dNTP mix
>
> d.       1.0µl of RNaseOUT™ (40 U/µl )

5.  Add the mixture to the annealed primer and RNA.

```{=html}
<!-- -->
```
5.  Add 2 µl of 400 U/µl SuperScript™ III RT and incubate at 48°C for
    2hrs . (Final volume is 30µl)

6.  Incubate at 70°C for 5 minutes to stop reaction.

7.  Cool down by spinning in a microcentrifuge at maximum speed for 1
    minute.

8.  Add 2μl of 2 U/μl RNase H and incubate at 37°C for 20 min.

9.  Add 0.5 μl of 0.5M, pH 8.0 EDTA, mix well and proceed with
    purification.

***II. [cDNA purification:]{.underline} *** (QIAGEN MINElute
purification kit)

1.  Add 200 μl of Binding buffer ***PB*** to each RT reaction and mix
    well.

> [Note]{.underline}: recommended maximum is 2 RT reactions per column.

2.  Apply each reaction to separate spin columns. Incubate for 1 minute.

```{=html}
<!-- -->
```
2.  Spin for 1 min at full speed.

3.  Discard flow-through.

4.  Add 500μl of Wash buffer ***PE*** per reaction (Be sure that ethanol
    was added to ***PE*** buffer).

5.  Spin for 1 min at full speed.

6.  Discard flow-through.

7.  Spin for 1 min at full speed to eliminate the possibility of
    carrying over Wash buffer.

8.  Place columns in a fresh 1.5ml microcentrifuge tubes.

9.  Add 10μl of 1:10 Elution buffer ***EB*** directly to the membrane.
    (dilute elution buffer 1:10 with Molecular Biology Grade water).

10. Incubate for 1 min. at room temperature.

11. Spin for 1 min at full speed.

12. Add another 10μl of diluted ***EB*** buffer to the membrane.

13. Incubate for 1 min. at room temperature.

14. Spin for 1 min at full speed.

15. Dry down in SpeedVac for \~15 min at medium temp.  DO NOT OVERDRY!

>   

***III. [NHS-ester containing dyes coupling reaction:]{.underline}***

1.  Resuspend cDNA pellet in 5µl of 2x coupling buffer.  (If pellet was
    over dried gently heat at 37° C for 15 minutes to aid in the
    resuspension process.)

```{=html}
<!-- -->
```
1.  The first time a tube of dye is used, resuspend in 45µl DMSO. Use
    DMSO provided with the kit.

2.  Add 5µl of the resuspended monofunctional reactive dye to cDNA.

3.  Mix thoroughly by gently pipetting up and down.

4.  Incubate minimum for 30 minutes at room temp in the dark, flicking
    the tubes occasionally.

>  
>
>  
>
>  
>
>  
>
>  

***IV. [Dye-Coupled cDNA Purification:]{.underline}*** *(using QIAGEN
MINElute purification kit)*

1.  Add 10μl of 3M Sodium Acetate, pH 5.2 to each labled cDNA reaction,
    mix well.

```{=html}
<!-- -->
```
1.  Add 200μl of Binding buffer ***PB*** to each reaction and mix well.

2.  Apply each reaction to a separate spin column.

3.  Incubate for 1 min. at room temperature.

4.  Spin for 1 min at full speed.

5.  Discard flow-through.

> 7.       Add 500μl of Wash buffer ***PE*** per reaction (Be sure that
> ethanol was added to ***PE*** buffer).

8.  Spin for 1 min at full speed.

```{=html}
<!-- -->
```
8.  Discard flow-through.

9.  Repeat wash step.

10. Discard flow-through.

11. Spin for 1 min at full speed to eliminate the possibility of
    carrying over Wash buffer.

12. Place columns in a fresh 1.5ml microcentrifuge tubes.

13. Add 10μl of 1:10 diluted Elution buffer ***EB*** directly to the
    membrane

14. Incubate for 1 min. at room temperature.

15. Spin for 1 min at full speed.

16.  Add another 10μl of 1:10 diluted Elution buffer ***EB*** directly
    to the membrane.

17. Incubate for 1 min. at room temperature.

18. Spin for 1 min at full speed.

19. Can read OD with the Nanodrop to determine labeling efficiency and
    cDNA concentrations.

 

***V. [Pre-hybridization:]{.underline}** (should start approximately 2
hours before setting up hybridization)*

Pre-hybridization buffer = 5X SSC, 0.1% SDS and 1% BSA. (Can make 10%
BSA stock and filter before use or purchase pre-filtered BSA; store
pre-hyb buffer at -20° C and thaw only once, warm to 42° C prior to
use.)

1.  Apply 80 μl of pre-hybridization buffer under a Lifterslip to the
    array and incubate for 42° C for at least 30 mins and up to 1 hour. 

```{=html}
<!-- -->
```
1.  Wash off the pre-hybridization solution by rapidly plunging the
    slide in distilled water for 2 mins, then transfer slide to 100%
    isopropanol for 2 mins.

2.  Allow slide to air dry completely prior to use or spin dry.

>  

***VI. [Setting up hybridization:]{.underline}***

1.  Combine Cy3 and Cy5 labeled targets together (\~20μl recovered for
    each).

```{=html}
<!-- -->
```
1.  Denature target at 100^o^C for 1 minute, then snap cool on ice.
    (Final volume should be about 40µl)

2.  Make fresh 2X Formamide hybridization buffer (50% formamide, 10x
    SSC, 0.2% SDS) and warm to 42^o^C just before adding to samples. 

3.  Add 20μl of water to wells in hybridization chamber to maintain
    humidity.

4.  Add 40μl of 2X F-hyb buffer to samples

5.  Load 80μl sample onto microarray under a Lifterslip.

6.  Incubate overnight (12-16 hours) at 42° C in water bath or
    hybridization oven.

 

**[Wash:]{.underline}  **

•          2x SSC, 0.1% SDS for 2 minutes, with occasional plunging

•          1x SSC, for 2 minutes, occasional plunging

•          0.2x SSC, for 2 minutes, occasional plunging

•          Spin 3 minutes / 650 rpm to dry

**  **

> **WASHES:**                     [2XSSC+0.1%SDS]{.underline}     
>              [1XSSC]{.underline}                 
> [0.2XSSC]{.underline}                
>
> [dH~2~0:]{.underline}                                  179 ml         
>           190 ml                    198 ml                      
>
> [20XSSC:]{.underline}                              20 ml           
>                          10 ml                         2 ml        
>               
>
> [20%SDS:]{.underline}                             1 ml       

#### *Scanning Arrays*

# Powering Up

The power switch is located on the left side of the scanner towards the
back. In newer scanners, the power switch is located on the power supply
connected to the scanner. Turn the scanner on (it can be used
immediately without warm-up) and use the mouse to double-click on
GenePix Pro icon. This opens the control software and initializes the
scanner. (NOTE: GenePix Pro *[demo]{.underline}* does not allow one to
save data; the scanner will not operate in the *[demo]{.underline}*
application.) If an error message appears indicating that the scanner
cannot be found the computer was probably started up with the scanner
turned off, so you can restart the computer with the scanner on. Once
the computer is on, the scanner can be turned on and off without the
need for restarting the computer.

# Inserting the Microarray

Once the scanner is on and initialized in GenePix Pro, the array can be
inserted. Open the slide port at the front of the scanner by gently
sliding the door to the left. Open the slide chamber by gently
depressing the thumb toggle and lifting up. Insert the slide [array side
down]{.underline} with the NCI array number/label towards the front. Be
sure that the slide is resting on all four edges of the slide track.
Lower the chamber top and gently roll the thumb toggle forward then back
-- locking the chamber down securely. Close the slide port door (you
should hear the scanner hum as it draws the chamber in).

# Scanning

The first step is to do a preview scan at low resolution to determine
the exact location of the array area. Click on the hardware settings
button in the bottom right hand panel on the screen to bring up the PMT
settings window. Within this window, 635 nm refers to the Cy5 channel
(red) and 532 nm refers to the Cy3 channel (green). Set the voltages at
about 700 for each channel (the maximum setting of 1000 is not
recommended) and set the *lines to average* field at 1. Another option
is to average more than one line, and people may do this to decrease the
electronic noise during high resolution scan. Next, click on the preview
scan button (top right). The large black rectangular field in the center
of the screen represents the slide area. As the machine scans in both
channels simultaneously, the combined fluorescence image will resolve in
the slide area. Be sure to utilize the brightness and contrast slide
controls at the left center of the screen. These levels can be adjusted
up and down using the mouse and only affect the slide image as you see
it on the screen -- not the actual image itself. Typically, the
hybridized area is easily observed. Once the scan has passed the
hybridized area, click on the red stop button (top right panel). Next,
use the view scan area tool (left center) to click-and-drag a box around
the array area that you want to scan. This box can then be moved and
adjusted using the mouse. If necessary, select the zoom mode magnifying
tool (at left center) to magnify the image and then adjust the box as
necessary. Zoom back out when finished by clicking on the undo zoom
button (top left).

After designating the area to be scanned, begin a 5 micron, high
resolution scan by clicking on the high resolution scan button (top
right). The ratio circle at the top left of the screen will be
automatically selected to allow you to see the combined fluorescence
image (e.g. red and green together). By selecting the wavelength 635 and
wavelength 532 circles, you can toggle between the two channels to view
the fluorescence in each channel separately. You should also adjust the
brightness and contrast settings to obtain optimal visualization of the
signals. Remember, the scanning process is dynamic and user-interactive.
The PMT settings, magnification tools, contrast/brightness settings, and
ratio/635/532 circles should be adjusted on the fly to rapidly determine
each PMT setting for maximal signal intensities and optimal
normalization. In general, this is accomplished by scanning 1/3 - 1/2 of
the array, adjusting the PMTs in the desired direction, and rescanning
as necessary. Ideally, adjustment of the PMTs up or down in each channel
achieves maximum signal intensities with minimal saturation (saturated
spots are colored white) and also balanced (normalized) intensities
between the two channels. In general practice, spot saturation should be
limited to \~1% of spots and with channels normalized the majority of
spots should appear approximately yellow (as viewed with the ratio
circle selected). You can also use the histogram to balance the two
channels, and attempt to make them overlap on the histogram chart.

Once the scanning is completed, the images must be saved. Array should
be rescanned without PMT changes before saving. Click on the save images
button at the right center. In the *Save Images* window: 1) name your
scan in the *File name* field, 2) set the *Save as type* field at
*Multi-image TIFF files*, 3) leave the *Naming* field blank (i.e. no
boxes checked), and 4) [check]{.underline} [both]{.underline}
*Wavelength 635 nm* and *Wavelength 532 nm* [boxes]{.underline} in the
*Images* field. The Cy-5 and Cy-3 TIFF images will be saved
simultaneously in a single file to the folder that you have specified.
Currently, writeable CDs, and 100 and 250 MB Zip disks ([all
IBM-formatted only]{.underline}) can be used on the ATC and 10/4B54 PCs
and are recommended.

It is very important to save your files to disk when you finish with the
scanner. The C and L drives \"fill up\" frequently and therefore must be
purged of all files by erasing the contents of the Data folder! So, make
sure to save your image files to disk before you leave the scanner. Once
you have saved your files, please drag any folders you created to the
recycle bin to help free up storage space. We periodically purge the
computers at the ATC of any files that are more than three months old.

#### *Array Analysis*

[GenePix Pro 5.0]{.underline} Microarray Analysis software was developed
by Axon Instruments. A detailed description and tutorial of the array
analysis process can be found in the GenePix Pro 5.0 User\'s Manual
which you can download from the following website as a PDF file (open
with Adobe Acrobat 3.0 or higher):
[http://nciarray.nci.nih.gov](http://nciarray.nci.nih.gov/)

The NCI Microarray Facility provides access to this software at two
locations: ATC/128 and 10/4B54. As of this time, GenePix can be run on
PCs only. You can purchase the GenePix software for your own lab if you
desire. This will facilitate analysis of your data and reduce the high
traffic flow on the facility computers designated for this purpose.

We recommend collecting your data on writeable CDs as this is the safest
storage media. For a few scans at a time data collection by Zip disk may
also be a viable option.

#### Overview

The goal of this analysis is to measure the intensity of Cy3 and Cy5
signals recorded in the scanned image(s) so that signal ratios (i.e.
expression ratios) can be calculated for each feature on the array. This
is accomplished in the following stepwise fashion: 1) image(s) are
opened in GenePix; 2) the appropriate GAL file ([G]{.underline}ene
[A]{.underline}rray [L]{.underline}ist) is uploaded; 3) grid blocks are
aligned on the feature blocks; 4) feature indicators are fitted to the
array spots; 5) measurements are performed; and 6) the Results file (a
.gpr file) is saved together with a color JPEG image (a .jpg file) to
your disk for deposition into the NCI Microarray Database
([http://nciarray.nci.nih.gov](http://nciarray.nci.nih.gov/) ).

#### 

#### GenePix Start-Up

Once GenePix Pro is open, click on the **Open/Save button** and select
*Open Images\...* (hot key Ctrl+O). This opens the \"Open Images\"
window. Browse the computer for your disk, select your image(s), and
then press the **Open button**. (NOTE: If your images were saved as
*single-image tiff files*, you will use the mouse to click on (i.e.
select) both files while holding down the shift key. Alternatively, if
the images were saved as a *multi-image tiff file*, you simply select
the file with the mouse.) A color image of your array will appear. Use
the magnification tool (hot key z) to zoom in on your array image. Click
on the **Open/Save button** again and select *Load Array List\...* (hot
key Alt+Y). This opens the \"Load Array List\" window. Browse for your
disk, select the appropriate *.gal file* (this stands for
[G]{.underline}ene [A]{.underline}rray [L]{.underline}ist and can be
downloaded at
[http://nciarray.nci.nih.gov](http://nciarray.nci.nih.gov/) ), and press
the **Open button**. You will be given two options: 1) \"replace
existing blocks and apply gene names\", and 2) \"apply gene names to
existing blocks\". Typically, you will select the first one (unless you
have saved a grid template from a previous analysis; this is discussed
below). The grid blocks will appear on top of the array image.

#### Gridding (block mode)

The goal here is to align the grid blocks with the array spots such that
each feature indicator (these are the circles that make up the grid
blocks) is relatively close to its corresponding spot. Then the *align
blocks* function will automatically fit each feature indicator to its
corresponding spot. There are two grid modes you need to be familiar
with - one is called \"block mode\" the other \"feature mode\". In block
mode, you use the mouse to manipulate the grid blocks. In feature mode,
you use the mouse to manipulate individual feature indicators. You can
select block mode by typing the hot key B, or right-clicking on the
mouse for the \"right-click menu\" from which you can select \"block
mode\". Similarly, you can select feature mode by typing the hot key F,
or right-clicking on the mouse for the \"right-click menu\" from which
you can select \"feature mode\". (NOTE: in block mode, the pointer tail
has a box on it; in feature mode, the pointer tail has a circle on it.)
In block mode, select the top left grid block and drag it over the block
of spots. Zoom in on the block. To align the feature indicators with the
spots, click on the corner and side boxes (i.e. small white boxes on the
perimeter of the grid block) and while holding down the mouse button,
drag the mouse to resize the grid block. (NOTE: you will get a good feel
for this with practice.) When you click on a corner, the opposite corner
is anchored. However, if you click on a corner and hold down the Ctrl
key, all three of the other corners are anchored allowing you to flex
the grid to your specifications. The grid blocks can also be moved using
the keyboard. Using the arrow keys the blocks can be moved in tiny
increments. Using the \"+\" and \"-\" keys on the number pad the blocks
can be tilted. Remember, you do not need to align each feature indicator
perfectly over each spot; just get them close and the auto-align
function you will use later will fit them perfectly. Once the first
block is aligned, hit the \"\>\" key to rapidly move to the next block
for aligning. Once all grid blocks are aligned, press the "Align Blocks"
button and select \"Align Features in All Blocks\". Or use the hot key
Shift+F5. The feature indicators will then automatically align to each
spot, resizing and repositioning as needed and flagging spots with very
low signals as \"Not Found\". The flagging feature is very useful in
that it allows us (once our data is in the database) to filter out
flagged spots thus reducing the noise (i.e. false positives) in the
data. (NOTE: by pressing the **options button** and selecting the
analysis tab, you can define the size limits of the spots that will be
measured; if the spot falls below or above the thresholds you set, it
will automatically flag it \"not found\". You can adjust this if
necessary depending on the degree of autoflagging that occurs.) After
the feature indicators are aligned, switch over to feature mode (by
pressing the hot key F) and move from block to block double-checking the
auto-align results.

**Gridding (feature mode)**

In feature mode, feature indicators can be nudged around and resized for
a modified fit if you desire, and flags can be removed and added as you
deem appropriate. To reposition a feature indicator, select the feature
indicator by clicking on it and use the arrow keys on the keyboard to
move it. To resize a feature indicator, hold down the Ctrl key and use
the same arrow keys. To remove or add flags, select the feature
indicator and right click to pull up the \"right-click menu\". Select
\"Clear Flags\" to remove a flag (hot key L) or select either \"Flag Not
Found\" or \"Flag Bad\" to add a flag. What\'s the difference between
\"Flag Not Found\" and \"Flag Bad\" . Both allow signal measurements to
be recorded for that feature, however, the \"bad\" flag tells you (when
you are doing higher-order analysis later) that the data for that spot
has been deemed artifactual by the person performing the analysis; the
\"not found\" flag tells you that the signals may not be reliable owing
to spots of anomalous size/shape or very faint signal. Generally
speaking, many users rely on the \"not found\" flag for tagging all
spots that appear unreliable. Using this simplified approach, we
consider the unflagged spots to be \"good\". Obviously, you are welcome
to use the flags as you wish. However, we recommend that you NOT use the
\"Flag Absent\" option as this results in [no data being
collected]{.underline} for that feature. The \"Flag Good\" option is not
yet in use as the database does not yet recognize this flag as a
selection criteria.

#### Measurements

Once you have visually inspected the feature indicators for good
placement, click on the **Analyze button** (hot key Alt+A). All
measurements are calculated at this step and are automatically displayed
in the \"Results\" tab. This represents your raw data and is formatted
as a *.gpr file*. [The *.gpr file* and a *.jpg file* (i.e. a color JPEG
image of the array) are the two files that must be saved and deposited
on the database for subsequent analysis.]{.underline} To save these two
files, press the **Save As\... button** to open the \"Save Results\"
window. Select your disk, name the file and BE SURE THE BOX AT THE
BOTTOM OF THIS WINDOW IS CHECKED so that the JPEG image is automatically
saved, too. Then press **Save** and the .gpr file and the .jpg file will
be saved to your disk.

#### GenePix Inspection Tools

#### To visually inspect your data, GenePix Pro employs a number of useful tools. Under the \"Image\" tab, you can place the pointer on a spot and see the gene name and intensity information in the feature viewer at the bottom left. Under the \"Scatter Plot\" tab, you can select spots for viewing using the feature viewer based on signal intensity or ratio by placing the pointer on a feature in the scatter plot. You can also select one or more features in the scatter plot and they will be highlighted in the \"Results\" file for viewing specific measurements. Under the \"Report\" tab, there are a number of scripts (with instructions) that allow you to distill the array data down to the most useful information. For example, the \"Interesting Genes Report\" allows you to set ratio cut-offs for generating an \"outlier\" list of the genes with the largest calibrated ratios (calibrated 3 different ways). This list can then be exported (saved) as a *.htm file*, which can be opened in Excel. Back at your lab, you can print out the outlier list and a picture of the array, which you can keep on file. For additional information, you can check out the Axon GenePix website at [www.axon.com](http://www.axon.com/) or contact Axon technical support at <tech@axon.com> .

[**Accessing the Database for Depositing Data and Using the Web Array
Tools**]{.underline}

Our informatics partners at CIT have recently begun teaching an
informatics training class designed to familiarize array users with the
NCI microarray database and the online analytical tools for higher-order
analysis. Once you have been trained by us at the ATC to set up hybs,
scan, and analyze the arrays you can attend the array informatics class
given by a CIT informatics representative. You should sign up for this
class through the CIT (CIT Training Course #972), and the signup page
can be directly accessed from the mAdb system home page
<http://nciarray.nci.nih.gov/> . During the class you will learn how to
access the database, deposit array data, and utilize the tools for data
interpretation and visualization (i.e. array-array comparisons,
hierarchical clustering, multidimensional scaling, and clone reporting).
Any future questions with concern only to the database can be emailed
to:

<madb-support@bimas.cit.nih.gov>.

With each array version that the facility prints, a new GAL file
([G]{.underline}ene [A]{.underline}rray [L]{.underline}ist - a list of
genes represented on the array that defines the relative position of
each element on the array) will be generated. The GAL file is uploaded
into the analysis software to identify the features on your array. The
naming of this file will clearly indicate which version of the arrays it
refers to. GAL files can be downloaded at the NCI/DCS Microarray
Database Gateway at
[http://nciarray.nci.nih.gov](http://nciarray.nci.nih.gov/). For any
questions you may have in the future, please feel free to email our
staff: <nciarrays-r@mail.nih.gov>.

**Building 10 NCI Array Center**

The Building 10 NCI Array Center houses two Axon GenePix scanners and
four computers in the facility, two set up for scanning and two set up
for analysis only. The PCs designated for analysis are equipped with
GenePix Pro 3.0 \-\--the same instrumentation/software that we provide
at the ATC for your training and subsequent use. There is no NOVELL
access from these computers, so you must bring a PC-Zip disk, or a
recordable CD to save your data. The center is located in 10/4B54 and is
maintained by the laboratory of Louis Staudt (NCI Metabolism Branch),
located around the corner from 4B54 in 4N114. The current procedure to
obtain access to the Array Center is to come by Bldg 10, Room 4N114 and
sign up on the clipboard on the left hand side as you walk in. This must
be done in person; they will not accept a phone call to sign you up.
Each researcher can sign up for up to two hours a day of scanning time.
You can access this key Monday - Friday from 9a.m. to 6p.m. with the
exception of Tuesdays and Wednesdays from 12:15 p.m. to 2:00 p.m. Ask
any of Dr. Staudt\'s lab personnel for the 4B54 key and sign-out pad.

You are also welcome to return to the main facility at the ATC (room
128) to set up your overnight hybs and wash, and/or just scan, and
analyze your arrays. Most of the previously trained researchers find it
convenient to hyb and wash their arrays in their own lab, and then carry
them to 10/4B54 or ATC/128 for scanning and analysis.

#### NCI-Frederick's LMT Microarray Lab

There are two Axon GenePix scanners housed at the LMT Microarray Lab.
This is located at 915 Tollhouse Road, suite 211, Frederick.

If you need to contact the NCI Microarray Center in Gaithersburg, you
may call 301-435-7888.

If you need to contact personnel at the Array Center in building 10, you
may call 301-496-8890.

If you need to contact personnel at LMT in Frederick, you may call
301-846-5676.

**[Ordering Arrays]{.underline}**

There is a website available to members of the NCI for ordering
microarrays and signing up for training. Just point your browser to -
[http:// arraytracker.nci.nih.gov /index.cfm]{.underline} The Microarray
Tracker website is the only mechanism for ordering arrays (human oligo
and mouse oligo) from the NCI Microarray Facility. Additionally, the
Microarray Tracker website is used to register for the Microarray
workshop.

Some of you may have set up an account when you signed up for training.
If you do not yet have an account on the Microarray Tracking System it
will be assumed that you have no microarray training and you will be
automatically signed up for the next class before you can order arrays.
If you are an experienced array user and do not need training you will
have to contact [nciarrays-r@mail.nih.gov]{.underline} to get
authorization to receive an account.

Please note that in order for our system to process your order your PI
must have an established Center Number in our database. A Center Number
is NOT a CAN number. In order to purchase microarrays on this ordering
system, users should first go to:
[http://arraytracker.nci.nih.gov/centercheck.cfm]{.underline} to check
if their PI has an established center number in our system. User\'s who
do not find their PI but do have the required information should go to:
[http://arraytracker.nci.nih.gov/centercheck2.cfm]{.underline} to fill
out the request form on that page. We will then notify your AO, and they
will be responsible for assigning a center number and replying to the
Array Tracker system. It will be the responsibility of your AO and PI to
insure that you have a valid Center Number.

If you have any problems or suggestions feel free to e-mail the NCI
Microarray Facility at <nciarrays-r@mail.nih.gov> . Microarrays are not
a stock item, please allow 3 - 4 weeks for printing and filling orders.
We are filling all the orders as quickly as possible, and you will be
notified by e-mail when your arrays are ready.

> *Rev 8/06*