Hybridization

5.1 Hybridize fluorescent cDNA to slide

1. Determine the volume of hybridization solution required. The rule of thumb is to use 0.033 µl for each mm2 of slide surface area covered by the cover slip used to cover the array. An array covered by a 24 mm by 50 mm coverslip will require 40 µl of hybridization solution. The volume of the hybridization solution is critical. When too little solution is used, it is difficult to seat the coverslip without introducing air bubbles over some portion of the arrayed ESTs, and the coverslip will not sit at a uniform distance from the slide. If the coverslip is bowed toward the slide in the center, there will be less labeled cDNA in that area and hybridization will be non-uniform. When too much volume is applied, the coverslip will move easily during handling, leading to misplacement relative to the arrayed ESTs, and non-hybridization in some areas of the array.

2. For a 40 µl hybridization, pool the Cy3 and Cy5 labeled cDNAs into a single 0.2 ml thinwall PCR tube and adjust the volume to 30 µl by either adding DEPC H2O, or removing water in a SpeedVac. If using a vacuum device to remove water, do not use high heat or heat lamps to accelerate evaporation. The fluorescent dyes could be degraded.

3. For a 40 µl hybridization combine the following components:

Arrays and samples can vary somewhat, making it necessary to vary the composition of the hybridization cocktail. In cases where there is residual hybridization to control repeat DNA samples on the array, more C0t-1 DNA can be used, as in the High Sample Blocking formulation. When there is diffuse background or a general haze on all of the array elements, more of the non-specific blocker components can be used, as in the High Array Blocking formulation.

4. Mix the components well by pipetting, heat at 98°C for 2 minutes in a PCR cycler, cool quickly to 25°C and add 0.6ul of 10% SDS.

5. Centrifuge for 5 min at 14,000 x g. The fluor labeled cDNAs have a tendency to form small, very fluorescent, aggregates which result in bright, punctate background on the array slide. Hard centrifugation will pellet these aggregates, allowing you to avoid introducing them to the array.

6. Apply the labeled cDNA to a 24 mm x 50 mm glass coverslip and then touch with the inverted microarray.

Applying the hybridization mix to the array and coverslipping it is an operation which requires some dexterity to get the positioning of the coverslip and the exclusion of air bubbles just right. It is helpful to practice this operation with buffer and plain slides before attempting actual samples. The hybridization solution is added to the coverslip first, since some aggregates of fluor remain in the solution and will bind to the first surface they touch.

7. Place the slide in a microarray hybridization chamber, add 5 µl of 3x SSC in the reservoir, if the chamber provides one, or at the scribed end of the slide and seal the chamber. Submerge the chamber in a 65°C water bath and allow the slide to hybridize for 16-20 hours.

There are a wide variety of commercial hybridization chambers. It is worthwhile to prepare a mock hybridization with a blank slide, load it in the chamber and incubate it to test for leaks, or drying of the hybridization fluid, either of which cause severe fluorescent noise on the array.

5.2 Wash off unbound fluorescent cDNA

8. Remove the hybridization chamber from the water bath, cool and carefully dry off. Unseal the chamber and remove the slide.

As there may be negative pressure in the chamber after cooling, it is necessary to remove water from around the seals so that it is not pulled into the chamber and onto the slide when the seals are loosened.

9. Place the slide, with the coverslip still affixed, into a Coplin jar filled with 0.5X SSC/0.01% SDS wash buffer. Allow the coverslip to fall from the slide and then remove the coverslip from the jar with a forceps. Allow the slide to wash for 2-5 minutes.

10. Transfer the slide to a fresh Coplin jar filled with 0.06X SSC. Allow the slide to wash for 2-5 minutes.

The sequence of washes may need to be adjusted to allow for more aggressive noise removal, depending on the source of the sample RNA. Useful variations are to add a first wash which is 0.5X SSC/0.1% SDS or to repeat the normal first wash twice.

11. Transfer the slide to a slide rack and centrifuge at low rpm (700-1000) for 3 minutes in a clinical centrifuge equipped with a horizontal rotor for microtiter plates.

If the slide is simply air dried, it frequently acquires a fluorescent haze. Centrifuging off the liquids results in a lower fluorescent background. As the rate of drying can be quite rapid, it is suggested that the slide be placed in the centrifuge immediately upon removal from the Coplin jar.