Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 130

Specifi c diseases are associated with a shift in the composition
of blood cells. Each class of leukocytes has its own gene expression
profi le, therefore isolating specifi c cells and determining the mRNA
content of those specifi c cells will result in the identifi cation of dif-
ferent gene profi les. However, examining the entire profi le (cumu-
lative effect of all cells present in the whole blood) has gained
interest as a novel technique for the identifi cation of biomarkers of
disease. By targeting the gene expression profi le of all peripheral
blood cell types, the entire signature for the disease of interest is
identifi ed and those transcripts with the highest expression (from
any cell type) are easily recognized. This is also the most clinically
useful approach, as a point of care test can be developed to deter-
mine the amount of specifi c RNA transcripts present in the whole
blood which can be used by nurses in the ER or medics in the fi eld,
compared to cell sorters or fl ow cytometry which requires trained
specialists.

Although peripheral blood samples are collected routinely in prac-
tice, it is relatively diffi cult to maintain the integrity and biological
signature of RNA since mRNA in the cytoplasm has an extremely
short half life. A widely used method for RNA extraction requires
the isolation of peripheral blood mononuclear cells (PBMC). This
additional step adds to both the time and instrumentation require-
ments for RNA isolation, and can potentially result in changes in
the biological signature of the RNA profi le ( 24 ).
A new technology that has recently gained entrance into the
clinical arena, especially clinical trials research is the Paxgene™
Blood RNA system. Paxgene blood RNA tubes contain a reagent
that protects RNA from degradation, preserving the RNA expres-
sion profi le during and after phlebotomy. Using Paxgene RNA
tubes eliminate the isolation of PBMCs by integrating and con-
solidating nucleic acid stabilization and RNA purifi cation thereby
reducing the unpredictability associated with RNA processing of
PBMC isolation and enhancing the accuracy of RNA expression
analysis. Paxgene RNA tubes are a reliable source of RNA isola-
tion and have been shown to be a valid approach to RNA extrac-
tion from human whole blood ( 25 ). Proof of principle studies
have been performed in our laboratory verifying the Paxgene
RNA isolation method (T.L. Barr, Unpublished data). For its ease
of use in the clinical setting, and its ability to maintain the specifi c
biological signatures at the time of the blood draw, Paxgene blood
RNA tubes (or Biosystems Tempus ® ™ Blood RNA Tubes) are
recommended.
Total RNA purifi ed using the Paxgene system is highly pure,
with A 260 / A 280 values between 1.8 and 2.2 and £1.0% ( w / w )
genomic DNA. RNA yields are typically ³3

2.1.1. Qiagen Paxgene™
Blood RNA System

g from 2.5 ml of
whole blood. It is recommended to follow manufacturer proce-
dure for highest yield and quality, although we have found the

μ