POPOÔ-3 Staining Protocol for Microarray Printing Quality

(Manuscript in preparation by Arpat B., Wilkins T.A.)

Materials

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 mM POPOÔ-3 in 5X SSC) (80 ml/slide)

1250 ml 5X SSC

0.25 ml 1mM POPOÔ-3 iodide (Molecular Probes, P-3584) in DMSO

Prepare in an Eppendorf tube freshlya

Supplies

Aluminum foil

Beckman, GPR Centrifuge (optional)

Forceps

Hydrophobic plastic cover slips (HybriSlipÔ, 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)

Toothpicks

ScanArray® 3000 (GSI-Lumonics)

Sterile Coplin jar

Sterile glass solution bottles

Method Steps

  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 ml of the staining buffer onto each slide. One 40 ml 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 coverslipe.
  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 the slide.
  4. Cover the humid chamber with aluminum foil to provide a dark environment during stainingf.
  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.

Notes

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-GAPSÔ coated slides (Corning, NY) can be used in the POPOÔ-3 Staining Protocol. Usually when CMT-GAPSÔ 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-GAPSÔ 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-GAPSÔ 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.

Commentary

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 some samples.

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 level.

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.

References

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

Updated: 06/20/00