Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 139

technology has developed exponentially from the use of manual dot
blots to robot generated complementary deoxyribonucleic acid
(cDNA) chips and oligonucleotide arrays. Recently, the technique
of constructing the arrays has been slightly modifi ed to detect single
nucleotide polymorphisms (SNPs) or copy number variations of
genes across different samples. This technique scans a number of
markers across the DNA to identify genetic variations associated
with disease and is typically employed in SNP GWAS. For both of
these techniques, the microarray can be custom designed to target-
specifi c molecular pathways (e.g., targeted analysis of cancer-related
genes or genes in an infl ammatory pathway).

The two largest companies manufacturing microarrays are
Affymetrix© and Illumina©. There are a number of other microar-
ray platforms available; however, the use of commercially devel-
oped products is encouraged to ensure robust and reproducible
results. In addition, the original spotted array technology for
measuring relative gene expression has been displaced by more
accurate platforms, such as the Affymetrix GeneChip and the Illumina
BeadChip that allows the determination of absolute gene expres-
sion levels. Gene expression microarrays generally fall into the
following categories: either cDNA or oligonucleotide. Classical 3¢-
based microarrays were designed with probes localized only to the
extreme 3¢ end of the gene; these assays depended on priming the
gene from the transcripts poly-A tail, which had its limitations.
Most of the newer chips from Affymetrix (Gene 1.0 ST array) and
Illumina (HumanHT-12 Expression BeadChip) have probes
distributed across the entire length of the gene, not just the poly-A
tail, providing more complete coverage. Automated sequencing
detection systems, have eliminated the need for radioactive label-
ing by labeling hybridized cRNA with fl uorescent dyes. In addi-
tion, the sensitivity of the microarray has dramatically increased
over the years; for example, the sensitivity of the Illumina BeadChip
is extremely high (1:250 K) since each gene is typically measured
about 30 times.
cDNA arrays contain long sequences of cDNA (~50 bases)
generated from gene libraries and amplifi ed using polymerase chain
reaction (PCR), which are then printed into spotted matrices onto
glass slides. Each spot corresponds to a specifi c gene or transcript
(probe). mRNA is extracted from the cells of interest and amplifi ed
using PCR in which two types of fl uorescent base pairs (Cy3 and
Cy5) are inserted into the generated cDNA. These fl uorescently
labeled cDNAs from both cell lines are then allowed to hybridize
to the glass chip with the cDNA transcripts generated from the
gene libraries. The amount of hybridization to the cDNA tran-
scripts on the glass chip is proportional to the amount of mRNA
expression in the cell and can be quantifi ed as a fold change in
expression between the two fl uorescent tags.

3.1.1. Basic Concepts
and Platforms