Biotech Glossary |
Bioinformatics |
Lab Protocol |
Notes |
Malaysia University |
HYBRIDIZATION OF FLUORESCENT cDNA PROBES TO DNA MICROARRAYS
Hybridization of Fluorescent cDNA Probes to DNA Microarrays
Overview
This protocol describes the hybridization of fluorescent cDNA probes to DNA microarrays. DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. After hybridization, non-specific and excess probe is washed away from the array. The fluorescence of each spot of the array will reflect the level of gene expression.
Procedure
A. Specialized Equipment
1. Hybridization Chamber: Custom made by Die-Tech or purchased from Corning Costar.
2. 22 X 22 mm cover slips.
3. Fine tipped forceps.
4. Slide rack: for 8 slides.
5. Slide chamber: Large enough to contain 200 ml of solution.
B. Hybridization of Fluorescent Probes to Microarrays
1. Resuspend the probe in a final volume of 12 to 15 μl in 4X SSC. Include competitor DNA as required (e.g., poly dA, CoT1 DNA for a human cDNA array; see Hint #1).
2. Place the array in the hybridization chamber.
3. Place 10 μl of 3X SSC on the edge of the slide to provide a humid local environment.
4. Add 0.3 μl of 10% (v/v) SDS to the probe.
5. Boil the probe for 2 min. While the probe is boiling, set aside several cover slips in preparation for the next steps.
6. Carefully pipette the probe onto the array without introducing air bubbles.
7. Immediately place a coverslip over the array with forceps, again avoiding air bubbles (see Hint #2).
8. Close the hybridization chamber and submerge it in a waterbath set to 65°C.
9. Incubate the hybridization for 4 to 24 hr.
10. Prepare the Wash Solution 1, 2, and 3.
11. Pour each Wash Solution into a separate slide chamber (three total) and place a slide rack into both slide chambers containing Wash Solution 1 and Wash Solution 2.
12. Disassemble the hybridization chamber and quickly transfer the array to the slide chamber of Wash Solution 1 (see Hint #3).
13. Wait until the cover slip slides off.
14. Gently plunge the slide rack in Wash Solution 1 several times (see Hint #4).
15. Transfer the array to the slide rack in Wash Solution 2 and gently plunge the rack several times.
16. Transfer the slide rack from Wash Solution 2 to Wash Solution 3 and gently plunge several times (see Hint #5).
17. Transfer the rack to a tabletop centrifuge fitted with microtiter plate buckets containing paper towels at the bottom. Balance the centrifuge and centrifuge for 5 min at 500 rpm.
18. Transfer the array to a fluorescent array scanner and scan the array immediately (see Hint #6).
Solutions
Wash Solution 3
0.05X SSC
Wash Solution 2
0.2X SSC
Wash Solution 1
1X SSC
0.03% SDS
SSC (20X)
pH 7.2
3 M NaCl
0.3 M Sodium Citrate
BioReagents and Chemicals
NaCl
Sodium Citrate
Protocol Hints
1. This step requires empirical determination. See Protocol ID #2254 and Protocol ID#2217 for starting conditions.
2. The contributor of this protocol recommends that this step be rehearsed several times with 10 μl of ddH2O on blank microscope slides to perfect the procedure.
3. If the array is exposed to air (i.e., the cover slip falls off before submersion into Wash Solution 1), a higher background of fluorescence may result.
4. Be careful not to scratch the array against the loose cover slip at the bottom of the chamber.
5. Removal of SDS is critical for meaningful results.
6. The fluorescent Cy5 dye bleaches during scanning. The contributor notes the use of the GenePix 4000A Scanner. Samples can be scanned with this scanner two days post hybridization instead of right away.
Citation and/or Web Resources
1. Lab website
