Hybridization and detection experiments with the HC ExpressArray Kit
Hybridization and detection experiments with the HC ExpressArray Kit
This experiment describes the method of hybridization and detection using HC ExpressArray Kit. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Hybridization and detection experiments with the HC ExpressArray Kit
Materials and Instruments
Antibody Dilution Buffer Hybridization Buffer Enhancement Buffer PBS PBST Tween 20 Wash Buffer Antibody Move I. Materials For more product details, please visit Aladdin Scientific website.
Centrifuges and rotary heads Fluorescence microarray scanners Microarray chips Water baths
1. Buffers, solutions and reagents
Antibody dilution buffer ~Kbr_~H~M~2~1~Kbr_~H~M~2~1 Hybridization buffer ~Kbr_~H~M~2~1~Kbr_~H~M~2~1 Enhancement buffer
Take 50 ml of Enhancement Buffer Concentrate ~A containing sodium azide), add to 950 ml of deionized water and mix vigorously for 10s.
PBS (137 mmol/L NaCl, 2.7 mmol/L KC1, 4.3 mmol/L Na2HP04-7H20, 1.4 mmol/L KH2P04, pH ~7.2)
PBST
Take 500ul Tween 20, add it to 1LPBS and mix vigorously for 10s.
Tween 20
Wash Buffer
Take 50 ml of Wash Buffer Concentrate ~A (containing sodium azide), add to 1.45L of deionized water and mix vigorously for 10s.
2. Antibody
Primary antibody ~Kbr_~H~M~2~1~Kbr_~H~M~2~1 Secondary antibody
Goat serum (contains sodium azide)
3. Centrifuge and rotor head
Centrifuges with bucket-type rotating heads
4. Specialized equipment
Fluorescence microarray scanner with an excitation wavelength of 532 nm (to detect Cy3)
Conical tube, 50 ml (e.g., VWR)
Tweezers
Hybridization cassette (e.g., Coming)
LiherSlips (Erie Scientific) or equivalent
Paper towels
Microarray chips on glass media consisting of cDNA, PCR products, or oligonucleotides 70 bp or longer
Water bath or humidified incubator, preset at 37°C, 55°C, and 75°undefined*.
5. Additional equipment
HC Express Avray Kit (Digene Corporation)
~undefined supplied by HC Express Array Kit (Digene).
~undefined See step 8.
II. Methods
1. Place the printed microarray chip into the hybridization cassette with the spot sample side up.
2. Carefully place the LifterSlip on top of the dot sample area.
3. Add 10-20ul of water to each humidification hole of the hybridization cassette.
4. In a centrifuge tube, add 1~25ul of total RNA (1~20ug) to 25ul of Hybridization Buffer provided in the kit, add H20 to bring the volume up to 50ul, vortex for 10s and mix the hybridization mixture.
5. Incubate the hybridization mixture at 95°C for 2 min to denature it.
6. Immediately centrifuge the tube at >8000 g for 5s.
7. Quickly pipette the Hybridization Mix onto the edge of the LifterSlip on the glass-breaking microarray and the mix will be evenly distributed on the array.
8. Seal the hybridization cassette and incubate for 3 h at 75°C in a water bath or lake-humidified incubator.
For cDNA arrays, 75°C is recommended For oligonucleotide arrays, 65°C should be used.
9. Remove the hybridization cassettes from the incubator and let stand at room temperature for 5 min.
10. Place the chip and LifterSlip into a 50 ml conical tube containing 35 ml PBST until the LifterSlip falls out.
11. Remove the chip and transfer to another conical tube containing 35 ml PBST and wash the chip for l0s by shaking, in a vertical motion.
12. Using tweezers, place the chip spotting side up on a paper towel to dry for 1 min. return the chip to the hybridization cassette and place a new LifterSlip above the spotting area.
13. Take 5ul of Primary Antibody and add to 45ul of Antibody Dilution Buffer to make Primary Antibody Staining Solution.
14. Pipette the Primary Antibody Staining Solution onto the edge of the LifterSlip on the broken glass microarray and it will be evenly distributed on the array.
15. Seal the hybridization cassette and incubate in a humidified incubator at 37°C for 1h.
After this incubation step, it is not necessary to return the hybridization cassette to room temperature to proceed to the washing step.
16. Repeat steps 10-12 with a new 50 ml conical tube and new PBST buffer.
17. Prepare secondary antibody stain by adding 5ul of goat serum and 2ul of secondary antibody to 43ul of PBST buffer.
18. Pipette the Secondary Antibody Stain onto the edge of the LiherSlip on the glass microarray and it will be evenly distributed on the array.
19. seal the hybridization cassette and incubate in a humidified incubator at 37°C for an additional lh. preheat the enhancement buffer to 55°C during incubation.
20. Place the chip and LifterSlip into a 50 ml conical tube containing 35 ml of Wash Buffer until the LifterSlip falls out.
21. Transfer the chip to another conical tube containing 35 ml PBST and wash the chip for l0s by shaking, in a vertical motion.
22. Transfer the chip to a third conical tube containing 35 ml of pre-warmed Enhancement Buffer and wash for another l0s.
Extending the wash time beyond 10s will reduce the signal.
23. Transfer the chip to a fourth conical tube containing 35 ml of wash buffer and wash for 10s.
24. Finally, transfer the chip to a dry 50 ml conical tube and centrifuge at 100-200 g for 5-7 min or until dry.
25. Scan the chip on a microarray scanner and analyze the data.