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Chapter 10: Nucleic Acid Platform Technologies
Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Blocking Polylysines on Homemade Microarrays
(Protocol summary only for purposes of this preview site)Homemade microarrays are printed on polylysine-coated slides. The lysines form a positively charged surface that can bind nonspecifically to the acidic nucleic acids during hybridization, resulting in significant background fluorescence. Thus, a key step in microarray processing is blocking all of the surface lysines not associated with the oligonucleotides in the microarray spots. The -amino group of lysine is succinylated by reacting with succinic anhydride (Fig. 1). Because anhydrides readily hydrolyze in water, use only fresh reagents that have not had the opportunity to absorb much water.
Protocol 9: Blocking Polylysines on Homemade Microarrays doi10.1101/molclon.000187Homemade microarrays are printed on polylysine-coated slides. The lysines form a positively charged surface that can bind nonspecifically to the acidic nucleic acids during hybridization, resulting in significant background fluorescence. Thus, a key step in microarray processing is blocking all of the surface lysines not associated with the oligonucleotides in the microarray spots. The -amino group of lysine is succinylated by reacting with succinic anhydride (Fig. 1). Because anhydrides readily hydrolyze in water, use only fresh reagents that have not had the opportunity to absorb much water.
The procedure is straightforward. Microarrays are, if necessary, rehydrated; excess liquid is removed by drying at a moderate temperature; and the succinylation reaction is performed. After the reaction is complete, the slides are washed and dried with ethanol, at which point they are ready for hybridization or they can be stored in a desiccator. Rehydration is necessary if the microarrays were desiccated after printing (see Protocol 1). When microarrays are stored in a desiccator, the spots typically dry out to form rings. Rehydration is important to restore the full spots.
It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.
- Ethanol (95) (see Step 11)
- 1-Methyl-2-pyrrolidinone
- Use only HPLC grade. If it has become slightly yellowed, then it is no longer usable, having absorbed excess water.
- Microarrays printed onto glass slides (either homemade [Protocol 1] or purchased)
- Sodium borate (1 M, pH 8.0)
- SSC (0.5) <A>
- Succinic anhydride
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The stock bottle of solid succinic anhydride should be stored under desiccation and vacuum (or under nitrogen).
Do not use if exposed to moisture!
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The stock bottle of solid succinic anhydride should be stored under desiccation and vacuum (or under nitrogen).
- Beakers (500 mL and 4 L)
- Centrifuge, fitted with microtiter plate carrier
- Glass-etching pen
- Glass slide racks and wash chambers (e.g., Thermo Scientific Fisher, catalog no. NC9516192)
- Gloves, powder-free
- Heat block set at 80C (see Step 5)
- Heating plate set at 80C
- Humid chamber, for standard-size glass slides (e.g., Sigma-Aldrich, catalog no. H6644)
- Microarray slide box, plastic
- Orbital shaker
- Stir bar that fits a 500-mL beaker
- Water bath set at 80C
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1.
Select 15 slides for postprocessing. Handle all slides with powder-free gloves. Determine the correct orientation of each slide. With a glass-etching pen, lightly mark the boundaries of the array on the backside of the slide.
- Marking the array boundary is important because after processing, the arrays will not be visible.
- 2. Add enough distilled water to a large 4-L beaker so that a slide rack will be completely submerged when placed inside. Place the beaker on a heating plate and heat to 80C.
- 3. Fill the bottom of a humidifying chamber with 0.5 SSC. Suspend the arrays face-up over the 0.5 SSC, and cover the chamber with a lid.
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4.
Rehydrate until all of the microarray spots glisten (usually 15 min at room temperature).
- Allow the spots to swell slightly, but do not let them run into each other. Insufficient hydration results in less DNA bound within a spot, and too much hydration will cause spots to run together.
- 5. Dry each array by placing each slide, with the DNA side facing up, for 3 sec on an inverted heat block set at 80C.
- 6. Place the arrays in a slide rack. Place the slide rack into an empty slide chamber that is sitting on an orbital shaker.
- 7. Prepare the blocking solution by measuring 335 mL of 1-methyl-2-pyrrolidinone into a clean, dry 500-mL beaker. Dissolve 5.5 g of succinic anhydride using a stir bar.
- 8. Immediately after the succinic anhydride dissolves, mix in 15 mL of 1 M sodium borate (pH 8.0). Quickly pour the buffered blocking solution into a clean, dry glass slide dish.
- 9. Plunge the slides rapidly into the blocking solution, and vigorously shake the slide rack manually, keeping the slides submerged at all times. After 30 sec, place a lid on the glass box, and shake gently on the orbital shaker for 15 min.
- 10. Drain excess blocking solution from the slides for 5 sec. Submerge the slide rack into an 80C water bath. Gently swish the rack back and forth under the water for a few seconds. Incubate for 60 sec.
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11.
Quickly transfer the rack to a glass dish of 95 ethanol, and plunge to mix.
- Make sure that the ethanol is crystal clear. Do not use if it contains particulates or is cloudy.
- 12. Take the entire glass dish with slide rack still submerged to a bench-top centrifuge. (Be sure to have an equivalently loaded slide rack ready to serve as a balance in the centrifuge.) Drain excess ethanol off the slides for 5 sec. Quickly, place the slide rack onto a microtiter plate carrier, and centrifuge in a bench-top centrifuge for 3 min at 550 rpm.
- 13. After centrifugation, the slides should be clean and dry. Remove the slides from the rack and store them in a plastic microscope slide box. Arrays may be used immediately, or can be stored for at least 34 mo in a desiccator at room temperature.
| Simpson RJ. . Year: 2003. Peptide mapping and sequence analysis of gel-resolved proteins. In Proteins and proteomics, pp. 343424. . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
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