Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 128

analysis of brain tissue and peripheral blood has identifi ed novel
candidates for diagnostics and therapeutics as well as disease patho-
genesis in the context of cerebral ischemia, trauma, and other acute
CNS injuries ( 1-4 ).
The study of gene expression profi les that correlate with DNA
variation is a powerful and complementary approach to the detection
of novel disease risk loci. Genome-wide association studies (GWAS)
are critical in providing an unbiased assessment of genes contribut-
ing to disease risk and/or response to the treatment. However,
there is a substantial gap between SNP association in a GWA study
and the understanding of how a genetic locus contributes to dis-
ease. Variation in gene expression can provide a more comprehen-
sive assessment of susceptibility to complex disease and is directly
modifi ed by polymorphisms in regulatory elements. Thus, gene
expression profi ling can provide immediate insights into the probable
biological basis for the disease associations found in GWAS and
identify networks of genes involved in disease pathogenesis ( 5, 6 ).
The physiologic response to acute brain injury is complex and
these methods are expected to remain increasingly important to
validate diagnostic and prognostic signatures of disease as well as
support pharmacogenomic approaches to novel treatment strate-
gies. Recent studies have demonstrated that specifi c patterns of
gene expression are found in different types of brain injury ( 7-9 )
and the profi les obtained via whole blood RNA analysis share some
congruence with the RNA profi le of brain tissue ( 10, 11 ). The
isolation of high quality mRNA, which most closely refl ects biology
at the time of collection, is of upmost importance for each of these
applications. The essential components of reliable gene expression
profi ling is the protection of mRNA from ubiquitous RNases and
prevention of transcriptional induction postharvest to ensure high
quality sample and usability for all downstream applications.
2. mRNA Isolation
and Purifi cation
Most mRNA isolation procedures begin with immediate cell lysis
via high concentrations of detergents and/or organic solvents.
Though effective, these traditional procedures are highly impracti-
cal for clinical use and can result in poor stabilization of RNA in
these conditions. The use of RNA later ® (Qiagen) as a stabilizing
reagent for tissue is gaining tremendous popularity as it allows for
convenient transportation at room temperature and easy processing
of large numbers of samples ( 12 ). In addition, the development of
automated and user friendly RNA isolation systems for whole
blood (e.g., Qiagen Paxgene™ Blood RNA System, Applied
Biosystems Tempus ® ™ Blood RNA Tubes) and brain (e.g., Qiagen
miRNeasy Mini Kit) has increased usability and quality of mRNA