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| 1 | 2008
Klinisk Biokemi i Norden
(Fortsat fra side 23)
has proven its usefulness in diagnosis. Quantification
of the kinetics in minimal residual disease such as
chronic myeloid leukemia or acute lymphoblastic
leukemia by RT-PCR has resulted in new clinical stag-
ing and response to treatment (18;19). Application
of microarray methodology on to carcinoma tissues
(breast, colon or prostate) has resulted in the discovery
of response changes in a variety of genes. Some of
these transcripts may potentially in the future serve
as biomarkers to detect occult tumor cells in blood
originating from solid cancers (18;20;21). Ma et al
suggest that peripheral blood cells undergo profound
molecular changes during atherogenesis showing 108
genes differently expressed in coronary artery disease
peripheral blood cells as compared to normal (22).
Microarray analysis performed on total RNA in blood
cells from patients with schizophrenia or bipolar dis-
orders have shown that each disease state exhibited
a uniquely expressed genome signature as compared
to normal (23). Whole blood is supposed to become
the most important clinical specimen also to obtain
surrogate markers for diseases that are not primarily
associated with peripheral blood (24-26)
Thus, blood derived RNA appears to represent
a novel alternative to tissue biopsies as a source for
mRNA gene expression profiling. However, given the
heterogenous blood cell population and the challenge
of isolation of high-quality total-RNA there are many
factors to be accounted for during sampling, analyzing
and evaluation of the results.
Preanalytical precautions
Intracellular RNA will be rapidly degraded ex vivo by
specific and nonspecific endonucleases if not stabilized
immediately upon sampling. This will lead to changes
in the gene expression profiles. Standardization of this
preanalytical step may now more easily be done by
using integrated and standardized systems for collec-
tion and stabilization of whole blood specimens such
as the PAXgene (Qiagen) or the Tempus (Applied
Biosystems) systems. These systems have been validat-
ed to maintain RNA in a satisfactory way both at room
temperature for 5 days and when kept frozen. One
technical obstacle using these systems however, is that
RNA from reticulocytes will be isolated at the same
time. Reticulocytes are transcriptionally inactive, but
contain huge amounts of globin mRNAs. The number
of reticulocytes are normally 10 fold that of leukocytes
and they will therefore contribute up to 70 % of the
total RNA from blood (26). These circumstances have
been shown to decrease the sensitivity of detection of
other transcripts in some of the detection methods, but
methods for reduction of globin mRNAs not affecting
further analysis, are now available (26).
In an epidemiological setting robotized isolation
of total RNA is a prerequisite. Conventional, manual
isolations methods are demanding and will be cumber-
some with a high number of samples. Lability of RNA
molecules and the numerous possibilities to pollute
samples during isolation procedures, forces the isolat-
ed total-RNA to be quality checked for the deleterious
effect of RNases before subjecting the RNA to further
analysis. This may now be tested for by running the
samples in a dedicated electrophoretic system (27).
Quantification of specific mRNA transcripts
Most methods for quantification of specific mRNA
transcripts require enzymatic steps in advance of
detection, such as reverse transcription, to synthesize
cDNA from mRNA or labeling of cRNA. Real time
PCR or microarrays are most often used as analytical
platforms for the quantification.
Gene expression profiles from blood cells can be
obtained either by analyzing the global gene expression
by microarray technology or by quantification sets of
specific transcripts by quantitative RT-PCR.
Global gene expression profiles may give informa-
tion on a large number of transcripts. Depending on
array type, filtering and study design genes overex-
pressed or suppressed may be registered active at a
given time. The level of expression of certain genes
may signify a particular disease state. Genes thus con-
sistently overexpressed or suppressed in a certain clini-
cal context may be considered as biomarkers. Many
diseases are polygenic and triggered by genetic, envi-
ronmental and physiological factors. Software clusters
of relevant genes may be performed to distinguish
among different disease pattern.
Quantitative RT-PCR is normally used for detec-
tion on known specific transcripts. Different analytical
platforms offer facilities for quantification of one to
384 transcripts at a time. This makes quantification
of gene expression profiles from clusters of genes pos-
sible within a short time and the future will probably
provide “cardio-card”, “diabetic-card”, “hypertension-
card”. The combination of microarray analysis, that
identify panels of genes relevant for a disease state,
