Using a miRNA microarray can be broken down into 5 steps:
1. Post-Processing (Can be done ahead of time)
2. Isolation of Small RNAs from sample (Can be done ahead of time)
3. Labeling of RNA (Done the day of hybridization)
4. Hybridization
5. Washing and Scanning (Done the day after hybridization)
Note: You will be applying RNA directly to the array, always use gloves when handling the arrays. Arrays should be stored desiccated at room temperature.
1. Post-Processing—After post-processing arrays can be used for up to several weeks
Materials
Metal slide racks
Re-hydration trays (Sigma H6644)
Centrifuge with slide rack adaptors
Diamond-tipped glass etching pen (VWR 52865-005)
StrataLinker for UV cross-linking
Protocol
a) Pour 100 ml 0.5x SSC into hydration tray and warm on slide warmer. Set the warmer to 37℃
b) Pre-heat a heating block at max (>100℃)
c) After processing, the arrays will not be visible, so their boundaries need to be marked. Holding the diamond pen perpendicular to the slide, mark the boundaries of the array on back side of the slide.
d) Set slide array side down on the hydration tray and observe spots until full hydration is achieved (this will look like a light layer of condensation covering the array). Do not rehydrate more than 1 min.
e) Upon reaching full hydration, dry the slides by flipping one at a time onto the heating block with the array face up. Do this in one smooth motion, with one hand, pinching the array at one end and flipping it over as you move it to the hot plate. Having a good bright light at the right angle will help you see the slide drying. The array should dry within 1-2 seconds. Remove the slide and place into a metal slide rack.
f) UV cross link the arrays at 60mJ (if you are using a stratalinker, push the energy button, lighting-up the indicator for ujoulesx100, enter 600 and then press start).
g) Measure 335 ml of 1-methyl-2-pyrrolidinone into a clean, dry 500mL beaker.Dissolve 5.5 g of succinic anhydride in the 1-methyl-2-pyrrolidinone using a stirbar. Note that the stock bottle of solid succinic anhydride should be stored under desiccation and vacuum. Do not use if exposed to moisture, avoid using clumps.
h) IMMEDIATELY after succinic anhydride dissolves, mix in 15 ml of 1M sodium borate pH 8.0.
i) Quickly pour the buffered blocking solution into a clean, dry glass slide dish. Plunge the slides rapidly into blocking solution and vigorously shake, keeping the tops of the slides under the level of solution. After 30 seconds of plunge-mixing,put a lid on the glass box, and let shake gently on a rotator for 15 minutes.
j) Transfer slide rack to a glass jar filled with room temp distilled water. Plunge the slides up and down a few times in the water.
k) Transfer the rack to a glass dish of 95% EtOH and plunge several times to rinse.Make sure the EtOH is crystal clear. Do not use if it appears to have particulates or appears cloudy.
l) Spin slide rack in a benchtop centrifuge for 1 minute at 550 rpm.
m) After spinning, the slides should be clean and dry (if not dry you can try spinning again, if not clean you can try re-washing in ethanol followed by another spin).
