2. Isolation of Small RNAs from sample—it is usually advisable to check for degradation of your RNA either after extraction or before this step. This can be done on a gel or on an Agilent Bioanalyzer.
There are two options for small RNA isolation. 1) flashPage, an Ambion product, which is quick but not efficient for running many samples in parallel, or 2) acrylamide gel size fractionation—slow, but many samples can be run in parallel.
1) Purify RNAs of ≤40 nt for use on the array using Ambion’s flashPAGE electrophoresis unit according to their protocol:
http://www.ambion.com/techlib/prot/bp_13100.pdf.
RNAs can then be recovered by the flashPage
(http://www.ambion.com/techlib/prot/bp_12200.pdf) clean-up kit or by ethanol precipitation (http://www.ambion.com/techlib/misc/acetate_precip.pdf) (Note: Do not use a glycogen carrier).
or
2) Purifying small RNA on a denaturing acrylimide gel
Materials
10% Denaturing Polyacrylamide Gel
Trizol Purified RNA Samples (50-500 ug)
Gel Loading Buffer (with bromophenol blue and xylene cyanol)
Ethanol
3 M Sodium Acetate
15 mL Falcon Tubes
Protocol
b) Mix the sample RNA with equal volume of gel loading buffer and heat at 70°C for 2 min, and put on ice. Rinse out urea with reservoir buffer.
c) Run the gel until there is about 1.5 cm separating the xylene cyanol and bromophenol blue bands.
d) Cut out the slice between the bands and cut apart separate samples. Use a syringe plunger to crush the gel. Place in a 15 mL falcon tube and add 1 mL of 0.3M sodium acetate for every cm in width of lane used to run sample.
e) Rock overnight at 4°C.
f) Centrifuge tube for 5 minutes (2000 xg), collect supernatant and put at -20°C.
g) Add to the gel 1/5 the volume of sodium acetate used for the first elution. Rock for 1 hour at room temperature.
h) Centrifuge as before, collect the supernatant and add to the supernatant from the first elution.
i) Add 4 volumes of 100% ethanol to the collected supernatant. Vortex.
j) Incubate at -20°C overnight.
k) Centrifuge at 16,000 xg at 4°C.
l) Discard supernatant.
m) Wash the pellet with 500 μL 75% ice cold ethanol. Centrifuge at 16,000 xg for
10 minutes at 4°C. Discard the supernatant and air dry the pellet.
3. Labeling of RNA—this is best done just before hybridization, although there is one possible stopping point in the protocol, after which the RNA can be stored for 1-2 days.
Label small RNAs using Ambion’s mirVANA labeling kit according to their protocol:
http://www.ambion.com/techlib/prot/fm_1562.pdf.
Note: The Cy3 and Cy5 fluorophores will photo bleach, it is best to minimize their exposure to light as much as possible—this includes the hybridized array.
