POPO-3 Staining Protocol for Microarray Printing Quality
(Manuscript in preparation by Arpat B., Wilkins T.A.)
Solutions and Reagents
Deionized dH2O (ddH2O)
20X SSC (3 M sodium chloride, 0.3 M sodium citrate)
175.3 g sodium chloride
88.2 g sodium citrate
Dissolve in 800 ml dH2O, adjust pH to 7.0 with 10 N sodium hydroxide
Adjust volume to 1 liter with dH2O, dispense in aliquots and sterilize by autoclaving
Humidifying Buffer (3X SSC)
150 ml 20X SSC
850 ml dH2O
Washing Buffer (5X SSC)
60 ml 20X SSC
180 ml ddH2O
Staining Buffer (0.2 M POPO-3 in 5X SSC) (80 l/slide)
1250 l 5X SSC
0.25 l 1mM POPO-3 iodide (Molecular Probes, P-3584) in DMSO
Prepare in an Eppendorf tube freshlya
Beckman, GPR Centrifuge (optional)
Hydrophobic plastic cover slips (HybriSlip, Grace BioLabs)
Plastic humid chamber (Sigma, H6644) orb All-purpose laboratory wrap
Glass or plastic body (e.g. Eppendorf tube rack)
Pyrex baking dish (2QT-2L, Corning, NY, USA)
ScanArray 3000 (GSI-Lumonics)
Sterile Coplin jar
Sterile glass solution bottles
1. Fill the humid chamberb half full with 3X SSC to provide humidity during the staining procedure. Put
the slide(s)c, d on the proper places printed side up.
2. Pipette 2 40 l of the staining buffer onto each slide. One 40 l should be applied towards one end of
the slide and the other towards the other end far as possible as from the arrays within the range of the
3. Immediatelya place the hydrophobic plastic coverslips. Preferentially put first one edge then slowly lay
down towards the opposite edge to avoid air bubbles from being introduced between the coverslip and
4. Cover the humid chamber with aluminum foil to provide a dark environment during staining f.
5. Stain at room temperature for 30 minutes.
6. Fill the Coplin jar with washing buffer. Plunge each slide into the washing buffer. Let the coverslip fall
down by agitating the slide and take the coverslip out of the buffer with the help of a forceps. Keep
each slide in the washing buffer for 10 seconds.
7. Dry slides completely before scanning by either centrifuging (Beckman, GPR Centrifuge) at 500 rpm
for 5 minutes or taking them quickly out of the washing buffer with an angle less than 90° between the
non-printed side of the slide and the buffer surface and placing with the same angle on a Kimwipe.
8. Scan the slides with ScanArray® 3000 (General Scanning) in the first channel (excitation at 543 nm,
emission for CY3) with settings at 80% laser power and 80% HV power supply.
a. The cationic dye, POPO-3, appears to be readily adsorbed out of aqueous solutions onto surfaces
(particularly glass). Therefore aqueous solutions of POPO-3, like the staining buffer, should not be
prepared in glassware and stored for long-term. For the same reason it is urged to use plastic cover
slips instead of glass cover slips.
b. Instead of plastic humid chambers a simple assembly can be constructed using a Pyrex baking dish
(2QT-2L, Corning, NY, USA). After filling the dish half with humidifying buffer, a glass or plastic
body (e.g. Eppendorf tube rack), which should not be taller than the pyrex baking dish, is placed into
the dish. The level of the buffer should not exceed the upper edge of the body used. Pairs of toothpicks
are placed on the surface of the table-like body. The paired toothpicks should be parallel and not more
than the length of a slide away from each other. Then the slide(s) are placed on the toothpick pairs
vertically with the printed sides up. After the application of the staining buffer (see Method Steps) the
dish should be wrapped with all-purpose laboratory wrap and covered with aluminum foil.
c. Both poly-L-lysine or CMT-GAPS coated slides (Corning, NY) can be used in the POPO-3
Staining Protocol. Usually when CMT-GAPS coated slides are used signal-background fluorescence
is increased. Also preliminary experiments indicated that poly-L-lysine coated slides older than 3-4
months are resulting in increased background activities. Similar effect did not observed with CMT-
GAPS coated slides. Some other experiments also indicated that it is possible to have very high
levels of background with some batches of home-made poly-L-lysine slides, whereas the same POPO-
3 buffer does not result in high background when used on CMT-GAPS slides.
d. POPO-3 is an intercalating cyanine dimer. The reported spectral characteristics of the dye which make
it possible to excite and detect the emission with the Cy3 channel of the microarray scanners are given
for dsDNA. Although the same spectral characteristics are expected for ssDNA/dye complexes,
changes in the dye:base ratio might shift the spectra. Therefore it is recommended to use it without
denaturing the DNA on the slide.
e. Although the slide surface is covered, POPO-3 can still be adsorbed out of the staining solution onto
the slide causing high background spots where the staining buffer was first applied. To overcome this
effect the staining buffer should not be applied directly onto the DNA arrays but instead far as possible
within the coverage of cover slips, and the slips should be placed as soon as possible after the
application of staining buffer.
f. Since the dye is photoactive the staining buffer and the stained slides should be avoided from light
exposure as much as possible.
Examples of the Method. The POPO-3 Staining is mainly developed to screen the quality of the
microarray printings. Generally two types of defects can be formed during microarray printings. One is the
depletion of some samples due to vaporization or leakage in the plate; the other is the inconsistent
deposition of the same sample though the whole set. The latter is mostly observed in the first or last slides
of a printing set; and sometimes researchers prefer to discard those slides. To decide whether it is the case,
slides from the initial, middle, and the last portion of one printing set can be screened for their quality using
the POPO-3 test. This also clearly indicates whether any printing defect has occurred due to depletion of
Normally in microarray analysis the amount of the DNA spotted on the slide is not the limiting step for the
hybridization, but due to experimental errors it is possible to have less DNA than required for saturation
which results in a less intense signal. In such cases the result might be interpreted in terms of low
expression. To avoid such misleading analyses the POPO-3 test provides a powerful control at the DNA
Time Considerations. Setting up the staining environment and preparation of the buffers requires
approximately 30 minutes. Depending on the number of slides the application of the stain, the staining and
the washing steps require approximately 50 minutes. The time requirement for scanning and analyzing the
images depends on the equipment used for this step, but usually it takes 30 minutes.
1. Rye, H.S, S. Yue, D.E. Wemmer, M.A. Quesada, R.P. Haugland, R.A. Mathies and A.N. Glazer. Stable
fluorescent complexes of double-stranded DNA with bis-interacting asymmetric cyanine dyes: properties
and applications. 1992 Nuc Acid Res 20:2803-2812