DRL Lab, 1-2
Indirect RNA Fragmentation and Labeling Protocol
Wipe off the RNA benchtop and your gloves with RNase away or similar product before
and after use. Make the following solutions with filtered RNase free tips/tubes/glassware:
DEPC water
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1ml DEPC/liter of DI water
Shake well.
Let stand for 24 hours.
Autoclave.
Transfer to small baked (180oC overnight) bottles for routine use.
5X Fe-EDTA complex
1. 7.5mM Ferrous Ammonium Sulphite (0.294 g)
2. 15mM EDTA (0.558 g)
3. Fill to 100ml w/ DEPC water
0.5M Ethylenediamine (EDA)
1. 0.15 g in 5ml DEPC water (RT, Filter sterilize)
Phosphate buffer pH 7.0
1. 39 ul phosphate buffer A (2.72g KH2PO4 in 100 ml DEPC water, filter
sterilized)
2. 61 ul phosphate buffer B (3.48g KH2PO4 in 100 ml DEPC water, filter
sterilized)
3. Fill with DEPC water to 200 ul.
0.3% Hydrogen peroxide w/w
1. Dilute 5 ul 30% w/w Hydrogen peroxide in 495 ul DEPC water.
2. Store at 4 C for 1 month.
1M Thiourea
1. 0.38g in 5ml DEPC water.
Sodium Carbonate Buffer pH 9.0
1. X g in 10 ml DEPC water
2. X ml 5M NaOH
3. Store at RT for 1 week
Monoreactive Cy-3 and Cy-5 dye concentrate
1. Resuspend one vial in 64 ul DEPC water
2. store at 4C for 1 month
3. reduce expose to light
Minimize exposure of dyes to light
Mix the following reactants in a 250 l eppendorf tube:
DRL Lab, 2-2
Ingredient
RNA
Fe-EDTA complex
Phosphate buffer
EDA
With DEPC water, fill to
100ul
10-20 ug
20ul
20ul
10ul
98.8 ul
Warm to 90oC for 3 minutes.
Add 1.2 ul 0.3% Hydrogen Peroxide.
Incubate at 90oC for 10 minutes.
Stop the reaction by adding 10 ul 1M thiourea.
Incubate at RT for 1 minute.
Wash with a microcon 30 concentrator using Sodium Carbonate Buffer.
a. 3X with 500ul solution
b. 14K spin for 15 min
7. Resuspend in 10 ul Sodium Carbonate Buffer.
8. Add 4 ul monoreactive cy-dye solution.
9. Incubate in the dark at RT for at least 1 hour.
10. Wash with the same microcon concentrator using DEPC water
a. 3X with 500ul solution
b. 14K spin for 15 min
11. Resuspend in 10 ul DEPC water and transfer to a fresh eppendorf tube.
12. Invert cup and recover solution by spinning down at low speed for 30s.
13. Measure dye incorporation using spectrophotometer and/or 20% polyacrylamide
gel.
14. Store at 4oC until ready to hybridize again an array.
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