NCI Microarray Manual

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##### 

**Introduction**

The use of microarrays for gene expression analysis has been gaining
acceptance in the biological community as a significant new tool. 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. 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. While the techniques used are based on Northern and
Southern blotting, thousands of gene sequences are detected
simultaneously rather than only a handful at a time examined with these
older methods.

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 of known sequence. The
experimental sample of RNA 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, we will hereafter 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 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 is the simplest method, 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 the poor quality or degraded state of the RNA in the
initial labeling reaction. In addition, 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 Probe 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                | Invitrogen          | 18021-071      |
+-------------------------------+---------------------+----------------+
| Poly (dA) 40-60               | Pharmacia           | 27-7988-01     |
+-------------------------------+---------------------+----------------+
| COT-1 DNA:                    | Invitrogen          | 15279011       |
|                               |                     |                |
| Human COT-1 DNA (for human    | Invitrogen          | 18440016       |
| arrays)                       |                     |                |
|                               |                     |                |
| Mouse COT-1 DNA (for mouse    |                     |                |
| arrays)                       |                     |                |
+-------------------------------+---------------------+----------------+
| Coverslips:                   | Erie Scientific     | 25X40I-2-4772  |
|                               |                     |                |
| 32-pin print: Lifterslips™    | Erie Scientific     | 25X40I-M-5227  |
| (25 X 40 mm)                  |                     |                |
|                               | Erie Scientific     | 25X60I-2-4789  |
| mSeries™ (25 X 40 mm)         |                     |                |
|                               | Erie Scientific     | 25X60I-M-5439  |
| 48-pin print: Lifterslips™    |                     |                |
| (25 X 60 mm)                  |                     |                |
|                               |                     |                |
| mSeries™ (25 X 60 mm)         |                     |                |
+-------------------------------+---------------------+----------------+
| Staining Dish/rack (10 slide) | Fisher              | 08-812         |
+-------------------------------+---------------------+----------------+
| 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., Inc. | AHC            |
| >                             |                     |                |
| > Single Hyb Chamber          | Corning             | 2551           |
| >                             |                     |                |
| > Single Hyb Chamber          | Telechem Int., Inc. | AHCXD          |
|                               |                     |                |
| Deeper hyb chamber to         |                     |                |
| 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 BSA | Invitrogen          | 15561-020      |
+-------------------------------+---------------------+----------------+
| 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.  Make dilution of 5.0 -- 20.0 µg of total RNA in 16.0µl of DEPC
    water.

2.  Add 2.0µl of 2.5μg/µl anchored oligo d(T)~20~ primer.

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

4.  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.

6.  Add 2µl of 400 U/µl SuperScript™ III RT and incubate at 48°C for
    2hrs.

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

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

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

10. 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 100 μl of Binding buffer ***PB*** to RT reactions and mix well.

> [Note]{.underline}: Can add a maximum of 2 reactions per column.

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

3.  Spin for 1 min at full speed.

4.  Discard flow-through.

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

6.  Spin for 1 min at full speed.

7.  Discard flow-through.

8.  Repeat wash step.

9.  Discard flow-through.

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

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

12. Add 10 μl of Elution buffer directly to the membrane. (20 μl of
    Elution buffer if 2 rxns were put on the column).

13. Incubate for 1 min.

14. Spin for 1 min at full speed.

15. Apply flow-through back on membrane.

16. Incubate for 1 min.

17. Spin for 1 min at full speed.

18. Discard columns, spec on Nanodrop to determine cDNA concentration.

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

*II. [Alternative procedure -- cDNA precipitation]{.underline}:*

1.  Add 3µl of 3M sodium acetate, pH 4.5.

2.  Add 1µl of 20mg/ml glycogen.

3.  Add 100µl of ice-cold 95% EtOH.

4.  Incubate at --20°C for at least 30 minutes. The reaction can be
    stored at this point for several days or up to 2 weeks.

5.  Spin the reaction at 13-14,000Xg for 20 minutes at 4°C. Carefully
    decant supernatant.

6.  Wash with 0.5 ml ice cold 70% EtOH and spin at 13-14,000Xg for 15
    minutes at 4°C. Carefully decant supernatant and let to air dry. A
    vacuum dryer can be used but 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.)

2.  The first time a tube of dye is used, resuspend in 45µl DMSO. Use
    DMSO provided with the kit.

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

4.  Mix thoroughly by gently pipetting up and down.

5.  Incubate for 30 minutes up to 1 hour 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 RT reaction, mix well.

2.  Add 100 μl of Binding buffer ***PB*** to RT reactions and mix well.

3.  Apply each RT reaction to separate spin columns.

4.  Incubate for 1 min.

5.  Spin for 1 min at full speed.

6.  Discard flow-through.

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

8.  Spin for 1 min at full speed.

9.  Discard flow-through.

10. Repeat wash step.

11. Discard flow-through.

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

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

14. Add 10 μl of Elution buffer directly to the membrane. (20 μl of
    Elution buffer for 48-pin print).

15. Incubate for 1 min.

16. Spin for 1 min at full speed.

17. Apply flow-through back on membrane.

18. Incubate for 1 min.

19. Spin for 1 min at full speed.

20. Can spec using 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 40 μl of pre-hybridization buffer to the array and incubate
    for 42° C for at least 30 mins and up to 1 hour. (If a 48-pin print,
    then apply 80 μl of pre-hyb buffer to the array.)

2.  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.

3.  Allow slide to air dry completely prior to use. (Can spin dry if in
    a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying
    before setting up hybridization.

## 

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

1.  Combine Cy3 and Cy5 labeled targets together (\~9 μl recovered for
    each).

2.  Add 1µl COT-1 DNA (8-10 µg/µl) and 1µl poly A (8-10 µg/µl).

3.  Denature target at 100^o^C for 1 minute, then snap cool on ice.
    (Final volume should be about 20µl.)

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

5.  Add 20μl of 3X SSC to wells in hyb chamber to maintain humidity.

6.  Add 20μl of 2X F-hyb buffer to samples. (Add 40 μl of 2X F-hyb
    buffer to samples for 48-pin print.)

7.  Load 40μl sample onto microarray. (Load 80 μl onto 48-pin print.)

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

Wash:

+-----------------------------------+----------------------------------+
| a\. **[cDNA                       | b[. **Oligo                      |
| slides:]{.underline}**            | slides:**]{.underline}           |
|                                   |                                  |
| -   2x SSC, 0.1% SDS for 2        | -   2x SSC, 0.1% SDS for 2       |
|     minutes, with occasional      |     minutes, with occasional     |
|                                   |                                  |
| > plunging                        | > plunging                       |
|                                   |                                  |
| -   1x SSC, for 2 minutes,        | -   1x SSC, for 2 minutes,       |
|     occasional plunging           |     occasional plunging          |
|                                   |                                  |
| -   0.2x SSC, for 2 minutes,      | -   0.2x SSC, for 2 minutes,     |
|     occasional plunging           |     occasional plunging          |
|                                   |                                  |
| -   0.05X SSC, for 1 minute       | -   [NOTE:]{.underline} The last |
|                                   |     wash of 0.05X SSC is omitted |
| -   Spin 3 minutes / 650 rpm to   |                                  |
|     dry                           | -   Spin 3 minutes / 650 rpm to  |
|                                   |     dry                          |
| ##                                |                                  |
+-----------------------------------+----------------------------------+

> **WASHES:** [2XSSC+0.1%SDS]{.underline} [1XSSC]{.underline}
> [0.2XSSC]{.underline} [0.05XSSC]{.underline}
>
> [dH~2~0:]{.underline} 179 ml 190 ml 198 ml 200 ml
>
> [20XSSC:]{.underline} 20 ml 10 ml 2 ml 0.5 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 facing 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 the performance of 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 is 1000 and
is usually not necessary) and set the *lines to average* field at 1.
Another option is to average more than one line, and people will 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 10 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. 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 C drive: Axon folder: Data folder
unless otherwise 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 and has been tailored to meet certain NCI needs. 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. We discourage the use of Jazz disks (IBM-formatted only)
as these are drives are not in every computer at the both ATC/128 and
10/4B54. For a few scans at a time data collection by Zip disk is also 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 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. (NOTE: if you plan on analyzing other arrays, you might
want to save this grid template so that you don\'t have to align grids
from scratch each time. To do this, use the \"Save Settings\" and \"Open
Settings\" selections under the Open/Save button.) 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, I 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 and the MTL
Laboratory. 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.

**FAQs**

### Who do I contact with questions? 

The best way to get a quick answer is to use the
<nciarrays-r@mail.nih.gov> email address. This is monitored daily and
any question will be answered as soon as possible by the appropriate
group or person.

If your question is about any array business (or Array Tracker) you
should always email <nciarrays-r@mail.nih.gov>.

If your question is particular to uploading data problems on the mAdb
website or to set up privileges you should contact
<madb-support@bimas.cit.nih.gov> .

**What are the microarrays composed of and how are they manufactured?**

Our microarrays are composed of 70-mer oligos immobilized on Corning
UltraGAPS™ coated slides. The oligos are spotted onto the slides using a
GeneMachines microarrayer. The arrayer can print up to 100 arrays at a
time. Arrays are typically printed in 32 or 48 sectors (or "blocks" of
spots) depending on the number of printing tips used. The printing tip
works much like a quill pen. It wicks up a fraction of a microliter of
oligo and deposits a fraction of a nanoliter of oligo onto each slide.
Each spot diameter can vary but averages \~100 um. Spots are printed
with center-to-center spacing ranging from 160-220 um depending on the
size of the array and density requirements. Printing with a 32-tip
configuration, it takes approximately 18 hours to manufacture 100
microarrays each consisting of 22464 spots for the Operon Human Version
2.0 oligo set. Printing with a 48-tip configuration, it approximately 22
hours to manufacture 100 microarrays each consisting of 34,580 spots for
the Operon Human Version 3.0 long oligo set. And printing with a 48-tip
configuration, it approximately 24 hours to manufacture 100 microarrays
each consisting of 38,467 spots for the Illumina Mouse MEEBO long oligo
set.

**How do the microarrays work?**

Fluor-labeled cDNA is generated from purified RNA (mRNA or total RNA).
One RNA sample is labeled with Cy3 (emits green fluorescence at 532 nm)
and the other is labeled with Cy5 (emits red fluorescence at 635 nm).
The labeled cDNAs are combined and hybridized to the same microarray.
The cDNA hybridizes to the probes via complementary interaction. The
ratio of Cy5 signal to Cy3 signal can be calculated for each spot on the
array and indicates the relative ratio of gene expression. These ratios
can be normalized and converted to color for visualization purposes.
Traditionally, a yellow spot indicates a 1.0 (or a 1:1) expression ratio
which means no difference in expression levels between the two samples
while a red spot (\>1.0) indicates that gene is expressed at higher
levels in the sample labeled with Cy5. Conversely, a green spot (\<1.0)
indicates that gene is expressed at higher levels in the sample labeled
with Cy3. (The expression ratio is generally reported as Cy5/Cy3, or red
over green.)

**How should the arrays be stored and how long do they last?**

The arrays at the NCI Microarray Facility are stored at room temperature
in a desiccant chamber for minimizing humidity. We recommend that the
slides be stored in slide mailers, at room temperature, in a sealed
container with desiccant to keep out dust and humidity. We have found
that the arrays stored this way last for 6 months and still deliver
optimal performance.

**Where do the genes represented on the microarrays come from?**

Currently, we are printing the Qiagen Human Version 2.0 set of oligos
for human arrays and Compugen Mouse Release 2.0 for mouse arrays. Each
company has their own proprietary bioinformatic algorithms for selection
of oligos. However, oligo design and optimization criteria include
incorporating alternative splicing structure by selecting probes from
regions that are common to a maximal number of predicted splice
variants, minimizing cross-homology, maintaining sequence quality by
avoiding sequencing errors and polymorphic sites, normalizing GC content
/ Melting Temperature across the collection, minimizing the distance
from the 3\' end, avoiding oligo secondary structures, and considering
genomic information.

**How can I find information for an oligo on the array?**

First go to the Qiagen's Oligo Microarray Database at
<http://oligos.qiagen.com/arrays/omad.php>. Follow the link for the
appropriate oligo set. Currently, we are printing Human Version 2.1 and
Version 3.0.

For mouse arrays information can be found at
<http://www.labonweb.com/chips/libraries.html> regarding the Compugen
Mouse Release 2.0 oligo library. For more information on the Mouse MEEBO
set visit
<http://www.illumina.com/products/dna/genomesets/meebo_mouse.ilmn>.

**How long can labeled cDNA be stored prior to hybridization?**

Some users have successfully kept labeled cDNA if stored at --20ºC under
dark conditions for up to one month. Store labeled cDNA without SSC, SDS
and before the final denaturation step.

**How soon after washing should the arrays be scanned?**

It is best to scan them as soon as they are washed. As a general rule,
the arrays should be scanned the same day they are washed -- preferably
within a few hours of washing. Be aware that during the summer months
when ozone levels are high, signal decay can occur quickly.

**How much total RNA is required for labeling?**

For maximum signal detection and reproducibility, we recommend at least
10μg of total RNA. The amount of total RNA required for labeling is
dependent on the quality of the RNA and the level of mRNA in the sample.

**How do I increase my hybridization signal?**

There are a number of ways to approach this question. If your signal is
consistently weak, try doubling the amount of total RNA used when
labeling. Often the mRNAs can account for a much smaller fraction of
your total RNA sample then expected. By increasing the amount of
template RNA used in the RT reaction, you can often increase the amount
of labeled target. However, it should be noted that this is not always
the solution. Degraded RNA is a problem that accounts for low signal as
well. In some tissue types, isolating high molecular weight RNA is
inherently difficult. If RNA quality is in question, we recommend
running a few micrograms out on a gel or using Agilent's Bioanalyzer to
check for degradation. In many cases, higher quality RNA is necessary
for better array hybridizations. Also, certain reagents that go into the
RT reaction can go bad overtime. DTT and dNTPs are particularly
sensitive to multiple freeze/thaw cycles. Fresh RT enzyme can sometimes
make a positive difference, too. New methods in probe labeling are
emerging that promise to increase signal sensitivity.

**How do I decrease the background signal on my array?**

Background often results from high salt and detergent concentrations
during the hybridization. Most often, high salt and detergent
concentrations occur as the result of evaporation of the hybridization
solution. Evaporation drives the salt and detergent concentrations up
and may facilitate the precipitation of target onto the glass surface
(particularly Cy5-labelled target). Evaporation can occur rapidly at two
particular steps in the protocol: 1) when the target is first pipetted
onto the array, and 2) following the hybridization when the hyb chamber
is opened and the array is exposed to cold, low-humidity air. Point
number 2 is potentially the most critical. Most hyb chambers have
aluminum bases. When they come out of the water bath, they remain hot
for several minutes. Once the cover is removed, the array is essentially
sitting on a hot plate in a cold environment. At this point evaporation
of the target around the edges of the coverslip can occur almost
instantaneously. Therefore, you must work quickly to remove the array
from the hyb chamber and submerge it in the first wash buffer.

Sometimes the background actually results from fluorescence emanating
from the opposite side of the array. We routinely clean the backside of
the arrays (after washing and drying) with moist breath and a Kim wipe
-- just prior to scanning.

In cases of particularly weak signal, we often have to scan at
near-maximum PMT voltage that results in relatively high background
levels. To combat this phenomenon, you should try to increase signal
(i.e. probe intensity) and scan at lower PMT voltage.

It has been reported that organics used during RNA extraction can
contaminate RNA samples and contribute to background fluorescence.
Likewise, benzene which is used to dehydrate 95% EtOH to 100% EtOH, has
been implicated in background artifacts. For this reason, many
researchers avoid using EtOH preparations made with 100% (200 proof)
EtOH.

We have seen that re-soaking the arrays in each wash buffer for several
minutes can reduce some forms of high background. Specific signal can
remain while background diminishes.

**How do I get rid of the "scars" that I occasionally see on my
arrays?**

Sometimes we observe symmetrical and parallel scratches on arrays that
would seem to indicate mechanical forces acting on the surface. The most
likely culprit is the coverslip. To avoid coverslip scratching, try a
more gentle approach when removing the coverslip. We recommend inverting
the array (coverslip down) in the first wash buffer (containing SDS
which helps loosen the coverslip) and gentle rocking up and down until
it starts to move. Then slowly tilt downward and let the coverslip fall
away on its own volition.

**How do I get rid of all those tiny "specks" of fluorescence I see on
my arrays?**

We call this speckling "schmutz" and it comes in a variety of flavors.
It can be particulate from the air (such as dust and lint fibers) that
settles onto the array surface or into the target during preparation and
binds to the array surface during the hybridization. It can be
precipitate such as SDS or cDNA aggregates that result from aberrant
salt and detergent concentrations or unknown contaminants. Sometimes
this phenomenon seems to be associated with a particular sample of RNA,
which can be corrected by RNA purification. If this phenomenon occurs
with frequency, you can clean the target on a 0.20-0.45 um filter, which
retains the particulates on the surface and allows the probe to pass
through.

**How do I minimize the "dim signal" areas on my array?**

The dim areas often have a circular boarder suggesting a bubble is the
culprit (although obvious bubbles do not always result in dim areas).
Though we try to minimize bubble formation when applying the coverslip,
bubbles that were not initially apparent can form during the
hybridization as a result of de-gassing. Sometimes the dim areas display
a pronounced signal gradient suggesting nonuniformity of hybridization
stringency across the surface of the array. We and others think that
this phenomenon arises due to initial volume gradients under the
coverslip. Volume gradients can result from warped coverslips and uneven
distribution of probe beneath the coverslip. When applying a standard
coverslip to the hybridization solution (pipetted onto the array
surface), we recommend a centered and level release such that the target
spreads beneath the coverslip in a radial fashion converging on all four
corners of the coverslip simultaneously.

Coverslips that possess "vertical lifts" on two or more edges can
potentially reduce volume gradients and eliminate dim areas.

**How do I just get better array results?**

Think critically about what you are doing when you are isolating your
RNA and making your probe. Quality is everything when it comes to
microarray science. Pure, high-molecular weight RNA is absolutely
required for good array results. Fresh reagents and meticulous technique
can make the difference between good probe and no probe. Be observant
every step along the way and always consider the molecular and physical
consequences with each manipulation of your RNA, labeled cDNA, and the
array or coverslip. Everyone has a learning curve, which usually begins
with consistently sub-average hyb outcomes and culminates in
consistently stellar hyb outcomes and array data. All it takes is a
little patience and a lot of critical thinking.

***Some Commercial RNA Amplification Kits:***

Many amplification kits/protocols have come to market within the past
year.

1.  Ambion <http://www.ambion.com/catalog/SubApps.html?4>

2.  Acrturus <http://www.arctur.com/research_portal/products/>

3.  Genisphere <http://www.genisphere.com/>

4.  Nugen <http://www.nugentechnologies.com/>

5.  Epicentre
    <http://www.epicentre.com/item.asp?ID=454&CatID=12&SubCatID=26>

Rev03-05