Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 140

Oligonucleotide arrays (aka. oligo arrays) consist of shorter
nucleotide sequences and have been pioneered by the commercial
companies Affymetrix© and Illumina© ( 38 ). Since the length of
the oligonucleotides is generally no longer than 25 bases, the density
of these chips is much greater than that of cDNA arrays, allowing
the user to assess greater numbers of gene products at the same
time. Oligonucleotide arrays can be manufactured as traditional
cDNA arrays, where the probes are spotted or synthesized on a
two-dimensional substrate or by using BeadArray technology.
Beadchips are constructed by introducing oligonucleotide bearing
3- m beads into microwells etched into the surface of a slide-sized

silicon substrate. Using the Illumina© platform, the beads self-
assemble onto the beadchips resulting in an average of 30-fold
redundancy of every full-length oligonucleotide. After random
bead assembly, 29-mer address sequences present on each bead are
used to map the array, identifying the location of each bead.
Oligonucleotide sequences are selected based on their uniqueness
to the target genes and may require the use of several matched
sequences for high specifi city to a single target. Similar to cDNA
arrays, mRNA is extracted from target cells and allowed to hybrid-
ize to the oligonucleotide array. However, with oligo arrays, only a
single fl uorescent channel is used, thus only a single sample can be
measured on one array.

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3.1.2. Procedures

Procedures for microarray analyses on most commercial platforms
generally contain the following and are slightly different from plat-
form to platform: RNA amplifi cation and cDNA synthesis, sample
labeling, hybridization, wash, stain, and signal detection. The user
must refer to guidelines provided by the manufacturer for specifi c
laboratory procedures.

(a) RNA amplifi cation and cRNA synthesis (sample labeling):
Begin with unlabeled total mRNA to reverse transcribe to
cDNA. This can be accomplished by available kits, such as the
Illumina TotalPrep RNA Amplifi cation kit to generate biotiny-
lated, amplifi ed RNA for hybridization to microarrays. The
procedure consists of reverse transcription with an oligo (dT)
primer (fi rst strand synthesis) and a reverse transcriptase
designed to produce higher yields of fi rst strand cDNA. The
cDNA undergoes a second strand synthesis and clean up to
become a template for in vitro transcription. The in vitro tran-
scription results in biotin-labeled antisense cRNA copies of
each mRNA in a sample which can be hybridized to the
microarray of interest.
(b) Hybridization, wash, and signal detection:
In this step, the labeled cRNA is applied to the microarrays,
hybridized overnight and washed prior to developing with a
fl uorophore, such as streptavidin-Cy3.