High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

DNA and RNA microarrays are very useful to study the interactions between nucleic acids and proteins, but they're also a convenient method for the preparation of sequence libraries. Photolithography allows for hundreds of thousands of unique sequences to be synthesized in parallel and is currently the only direct method for RNA synthesis on microarrays. The in situ photolithographic synthesis of microarrays can also be extended to chemically modified oligonucleotides, for instance with two prime fluoro of peptide nucleic acids.

Seeing the process of microarray fabrication and handling can help understanding how this complex machinery was developed from the well known standard solid phase DNA synthesis. Begin this procedure with microarray design and slide functionalization as described in the text protocol. Turn on the DNA synthesizer UV LED and its cooling fan.

Attach a UV intensity meter at the focal point of incoming UV light and turn it on. On the computer, start the WiCell Controller software. Turn on and initialize the micromere device.

Load an all white mask file by right clicking on DMD and then selecting load image. Right click on the UVS icon and select UV shutter open. Read the power value on the intensity meter and count 60 seconds.

After 60 seconds, read the power value again and note down the beginning and end values. Close the shutter by selecting UV shutter close and turn off the intensity meter. Calculate the average UV intensity value in milliwatts per centimeter square.

In the WiCell software, load the job, sequence, and protocol files in their respective sub-windows, then click send to send the sequence and protocol files to the DNA synthesizer. To assemble the synthesis cell place a thick perfluoroelastomer gasket on the quartz block of the cell. Then place a drilled, functionalized microscope slide on top of the first gasket, and verify that the holes on the slides connect with the inlet and outlet tubing of the synthesis cell.

Place a second, thin polytetrafluoroethylene gasket over the drilled slide, surrounding the two holes. Finally, place a second, functionalized but undrilled slide atop the second gasket. Now place a 4-screw metal frame on top of the assembled double-substrate cell Tighten the screw to the same clamping force using a torque screwdriver.

Attach the inlet and outlet tubing to the DNA synthesizer. Prime the acetonitrile wash line and verify the proper flow of acetonitrile through the substrates. Measure the volume of acetonitrile at the waste line after going through seven cycles of acetonitrile priming.

This volume should be two milliliters. Attach the synthesis cell at the focal plane of incoming UV light. In the case of library preparation, attach an extra inlet and outlet line to the back of the cell and fill the back chamber with the two milliliters of the betacarotene solution.

Start the synthesis by first clicking on Run in the WiCell software. At the first wait command in the job file, press start on the DNA synthesizer. After synthesis of regular microarrays, disconnect the cell from the synthesizer, and disassemble the cell.

Use a diamond pen to etch the synthesis number onto the glass slides. Etch the number on the non-synthesized face of each slide. Transfer the slides into 50 milliliter centrifuge tubes and store in a desiccated area until further use.

After the synthesis of library microarrays, first drain the betacarotene solution out of the chamber then wash the chamber twice by flowing five milliliters of methylene chloride through before draining. For DNA microarry deprotection, fill a staining glass jar with 20 milliliter of ethanol and 20 milliliter of EDA. Place the DNA-only microarrays vertically in the jar, close the lid, and leave the slides to deprotect for two hours at room temperature.

After two hours, retrieve the slides using tweezers and rinse them thoroughly with double-distilled water. Dry the slides in a microarray centrifuge for a few seconds before storing them in a desiccator. For RNA microarray deprotection, prepare a dry solution of 20 milliliters triethylamine and 30 milliliters acetonitrile in a 50 milliliter centrifuge tube.

Transfer one RNA microarray slide into the centrifuge tube, close the lid and wrap with plastic sealing film. Gently shake the centrifuge tube on an orbital shaker for one hour and 30 minutes at room temperature. Next, remove the slide and wash twice with 20 milliliters of dry acetonitrile before drying in a microarray centrifuge for a few seconds.

Following the first deprotection step transfer the RNA slide into the hydrazine hydrate solution, close the lid and wrap with plastic sealing film. After two hours of gently shaking on an orbital shaker, remove the slide and wash it twice with 20 milliliters of dry acetonitrile. Then dry in a microarray centrifuge for a few seconds.

If the RNA microarray also contains DNA nucleotides, proceed with a third deprotection step. Add the DNA/RNA microarray to a 50 milliliter tube containing a one to one EDA-ethanol solution After 5 min at room temperature, remove the slide, and wash the microarray twice with 20 milliliters of sterile water. Dry the slide in a microarray centrifuge and store in a desiccator.

In a 1.5 milliliter sterile microcentrifuge tube, prepare the hybridization buffer containing Cy3-labelled DNA, as described in the text protocol. Mix and vortex the solution. Carefully place a self-adhesive 300 microliter hybridization chamber over the synthesis area on each slide and pipet in the hybridization solution.

Cover the holes of the chamber with adhesive dots and wrap the entire slide in aluminum foil. Place the microarray slide into the hybridization oven, cover and let it gently rotate at the selected hybridization temperature for two hours. After two hours, detach the slide, remove the aluminum foil, pipette out the hybridization solution, and carefully tear off the hybridization chamber.

Transfer the slides into a centrifuge tube containing 30 milliliters of Non-Stringent Wash Buffer. Shake vigorously for two minutes at room temperature. Transfer the slide into a centrifuge tube containing 30 milliliter of Stringent Wash Buffer and shake vigorously for one minute.

Finally, transfer the slide into a centrifuge tube containing 30 milliliters of Final Wash Buffer. Shake for a few seconds. Dry the slide in a microarray centrifuge.

Now, place the dry microarray, synthesis area facing down, in the slide holder of the microarray scanner. To deprotect and cleave DNA libraries, immerse the slide into the cleavage solution, in a 50 milliliter centrifuge tube. Close the tube and wrap with plastic sealing film, before gently rotating in an orbital shaker for two hours at room temperature.

After two hours, remove the slide and wash twice with 20 milliliters of scrupulously dry acetonitrile before letting it air dry. With a pipette, apply 100 microliters of sterile water over the now discernible synthesis area. Pipet the solution up and down a few times before transferring it into a 1.5 milliliter microcentrifuge tube.

Repeat the process and combine the microarray eluate into the same tube. Evaporate the chip eluate to dryness and then redissolve into 10 microliters of nuclease-free H2O. Shown here are the results of a hybridization assay performed on a microarray containing the DNA and RNA versions of a 25mer sequence.

The scan in appears in a greenscale format corresponding to the excitation, emission spectrum of Cy3 fluorescence, with fluorescence intensity recorded in arbitrary units. There is significant variability in absolute fluorescence values between experiments. The results for three independent syntheses using the same fabrication parameters and the same post-synthetic handling are shown.

The 25mer DNA, when hybridized to its complementary Cy3-labelled DNA strand, will yield fluorescence signals ranging anywhere from 20, 000 to 30, 000, very rarely above or below. The 25mer RNA, when hybridized to the same Cy3-labelled DNA complement, will give fluorescence intensities on the corresponding features ranging from 15, 000 to 20, 000. However, fluorescence intensity of the RNA/DNA duplexes will occasionally drop below 8, 000, when the corresponding DNA/DNA duplexes will still fluoresce within the 20, 000 to 30, 000 range.

In such cases, the results for RNA may be regarded as sub-optimal. As with any other RNA-based experiment it is important to remember that RNA microarrays are sensitive to degradation, and should be handled under sterile conditions. Nucleic acid libraries collected from microarrays can be used in DNA or RNA sequencing as well as in the encoding of digital information on DNA.

The NED produces an intense UV light which should not be directly looked at. Because of this, it is advised to wear protective goggles while the instrument is in use.