4. Hybridization
The amount of RNA to use for hybridization will vary depending on the concentration of the miRNAs that you’re trying to visualize. Hybridizing with the small RNAs isolated from 20 μg of total RNA might be a good starting point.
Materials
Labeled RNA
Hybrization chamber
Waterbath at 42°C
Heat Block at 100°C
Ambion 3x hybridization buffer (Cat # 1567 mirVana™ miRNA Bioarray Essentials Kit)
22x25 mm lifter slips (Erie 22x25I-M-5226)
a) Heat 3x hyb buffer at 65℃ and vortex to resuspend
b) Add 5 to 10 100 μL drops of 5x SSC in the bottom of the hybridization chamber to prevent the arrays from drying out.
c) Place the slides in the hybridization chamber, array side up. Add the lifter slip over the arrays (this is where the diamond pen marking comes in handy). A flat metal spatula can be helpful to align one side of the lifter slip, so that you can gently drop the other side down. Use the spatula to align the lifter slip over the array. Be careful not to scratch the array surface.
d) Determine the volume of eluted RNA (should be around 20 μL) and add half this volume in hyb buffer (ie. for a 20 μl sample, add 10 μL).
e) Heat denature samples in a 95℃ heat block for 2 minutes. Microfuge to cool.
f) Immediately apply sample.
g) Close hybridization chamber, carefully transfer immediately to a 42℃ waterbath for 12-16 hours.
5. Washing and Scanning
Materials
Ambion Salt Concentrate (Cat # 1567 mirVana™ miRNA Bioarray Essentials Kit)
Ambion Detergent Concentrate (Cat # 1567 mirVana™ miRNA Bioarray Essentials Kit)
Metal slide rack
4 Glass slide boxes with covers (in which the slide racks fit)
Centrifuge rotor adaptors for metal slide racks
Protocols
a) Prepare 4 slide boxes with washing solutions. The first two are low stringency (4mL detergent concentrate, 20 mL salt concentrate, and 376 mL H2O) and the second two are high stringency (20 mL salt concentrate and 380 mL H2O) .
b) Carefully remove hybridization chamber from the water bath.
c) Remove slides one at a time from the chamber, tip off lifter slip in first low stringency bath and immediately place the slides in the metal rack sitting in the 2nd low stringency bath.
d) Wash slides in the low stringency bath, by plunging them up in down in the solution for1 minute (do not hit the bottom of the box too hard or you will damage your slides).
e) Wash in first high stringency bath for 1 minute.
f) Wash in second high stringency bath for 1 minute.
g) Spin slide rack in a benchtop centrifuge for 3 minutes at 800 rpm.
h) Place slides in a plastic slide box and scan immediately.
Note: Much of this protocol has only seen minor adaptations from protocols of Michael McManus and Caroline Mrejen, many thanks for their assistance
