Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 129

obtained in clinical samples, at the same time dramatically reducing
variability ( 13, 14 ). However, the RNA stabilization method may
have signifi cant infl uences on gene expression and reproducibility
and should be dependent on downstream application ( 15 ).

Since brain tissue is neither easily accessible nor practical to obtain
in clinical populations, there is an interest in identifying alternative
means by which gene expression is analyzed in most patients
affected by brain injury and neurological disease. Peripheral blood
is easily accessible in all clinical situations and therefore provides
the highest clinical utility. Several mechanisms have been proposed
to explain the genomic response of peripheral blood cells to cere-
bral injury. The most supported is that of the interaction between
peripheral immune cells and their ability to migrate and infi ltrate
compromised brain tissue ( 16 ). According to the clonal selection
theory, lymphocytes are genetically obligated to express unique
receptors or antibodies that when encountered by external anti-
gens respond by clonal expansion and differentiation ( 17, 18 ). In
addition, cells that respond to cerebral injury change their gene
expression in response to the environmental state of the brain ( 19 ).
Therefore, the cumulative changes in the transcriptional pattern of
blood could refl ect lymphocytes responding to cerebral events and
other whole blood components (such as red blood cells and plate-
lets) that contain a genomic fi ngerprint of the cerebral environ-
ment ( 20 ). Examining this, genomic fi ngerprint in the peripheral
whole blood can serve as an indirect surrogate for the genomic
response to trauma or ischemia within the brain. However, the
blood genomic fi ngerprint may also be altered by whole body
response to injury. Therefore, if this technique is to be used appro-
priately for the study of human CNS disease, it will be of primary
importance to accurately identify a control population to study the
genomic response to trauma and/or stress to ensure that the
genomic profi les identifi ed are specifi c for CNS injury and not
related to processes associated with trauma-related stress. This
approach could aid in earlier diagnosis of neurological disease,
identifi cation of targets for therapeutics, treatment decision making,
and predictions regarding prognosis ( 21, 22 ).
RNA isolated from whole blood typically consists of a high
proportion of mRNA encoding globin polypeptides. This high
proportion of globin mRNA may result in RNA labeling and
reduced sensitivity of hybridization on some microarray platforms.
A recent study demonstrated that globin reduction does not
increase the number of differentially expressed transcripts when
hybridizing to HumanRef-8 v2 BeadChips and therefore has little
impact on probe detection when using the Illumina platform ( 23 ).
However, this must be taken into consideration when using other
platforms and globin reduction should be considered if the goal of
the study is to identify rare or low expressed transcripts.

2.1. Total RNA
Extraction from
Peripheral Blood