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infer relative changes in expression between samples. Microarrays
are useful as they are able to screen large number of miRNAs
simultaneously on the basis of the ability of performing multiple
hybridizations in parallel with the oligo probes pre-spotted on a
chip [ 33 ]. However, microarrays are only semiquantitative and are
best suited to compare relative expression levels of miRNAs.
Therefore, microarrays can be used to screen for a range of miR-
NAs. On the other hand, another form of validation such as quan-
titative reverse transcription polymerase chain reaction (RT-qPCR)
is required to quantify expression. Furthermore, specificity can be
an issue in microarrays due to high degree of similarity between
closely related sequences of miRNA [ 34 ].
Before starting the experiment, RNA needs to be isolated from
the samples using a method that preserves small RNA species.
Subsequently microarray is performed in two steps including (1)
labeling and (2) hybridization.- Allow components of labeling kit to thaw on ice followed by
vortexing, and then spin them down using table microcentri-
fuge machine (see Note 3). - Combine the reagents included in the microRNA array label-
ing kit (e.g., Cy 5 or Cy3 according to the kit manual), spike-in
miRNA (3 μL of total RNA) in an RNase-free microcentrifuge
tube (see Note 4). - Pipette up and down several times to ensure all reagents have
been mixed thoroughly. - Incubate at 37 °C for 30 min then at 95 °C for 5 min followed
by a snap cooling on ice (see Note 5). - Briefly spin the sample and place back on ice.
- Combine this with 3 μL labeling buffer, 1 μL labeling enzyme,
and 2 μL dimethyl sulfoxide (DMSO) (or alternative according
to the kit manual). - Pipette up and down several times to ensure all reagents have
been mixed thoroughly. - Incubate at 16 °C for 120 min then at 65 °C for 15 min (see
Note 6). - Leave the reaction at 4 °C and perform hybridization on the
array within 1–2 h. - Adjust the volume of the labeled sample to 200 μL by adding
RNase-free water. - Add equal amount (200 μL) of 2× hybridization buffer to the
labeled sample (see Note 7). - Vortex and centrifuge to mix reagents.
3.2.1 Labeling
3.2.2 Hybridization
Moein Amin et al.