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| 1 | 2008
Klinisk Biokemi i Norden
(Fortsat fra side 26)
in years to come. Disease up- or down-regulated
genes that code for proteins that potentially may be
released into the blood stream, thus giving rise to
potential protein biomarkers, may become in focus.
As bioinformatics software tools become available
and more simplified, more disease specific infor-
mation will be mined. To what extent endogenous
siRNAs will add to the messengers remains elusive.
Obviously, the highly sophisticated and complicated,
intracellular RNA languages will need the cooperativ-
ity of several professions to advance the understand-
ing of systems biology.
As for plasma DNA and mRNA, new useful molec-
ular markers of cancer will appear. Their value
in monitoring or predicting disease, however, will
depend on increased basal understanding of how
these circulating nucleic acids are formed, and how
they enter (and leave) the plasma and other biological
compartment as well as well designed clinical studies
with well delineated clinical endpoints.
Methods
Isolation of nucleic acids from biological fluids
DNA is a stable molecule which easily can be isolated
from biological fluids such as whole blood, cerebros-
pinal fluid and tissues. RNA, however, may already on
sampling be degraded by RNases if not immediately
stabilized. With proper stabilization, DNA and RNA
molecules can be isolated both manually or robotized
from a cell lysate.
Available methods for isolation of nucleic acids
may be based on organic extraction or adherence
either to magnetic beads or columns. The method
of choice should document integrity and isolated
molecules should be representative for the content of
DNA or RNA in the cell lysates .
Reverse Transcription
Methods used for quantification of mRNAs can not
use mRNA molecules directly in their synthesis.
For quantification of specific transcripts a process
called reverse transcription has to synthesize cDNA
molecules with mRNA molecules as templates. This
provides a cDNA library synthesized from all the
mRNA molecules (the transcriptome) in the lysate
where furthermore PCR primers can pick the specific
target in the PCR reaction (49). Global gene expres-
sion profiles on micro array platforms are dependent
on mRNA molecules labeled with either fluorogenic
or biotinylated molecules. This involves several steps
before a sample can be hybridized to an array.
Real-time PCR
The Polymerase Chain Reaction is a laboratory tech-
nique that can amplify one molecule of cDNA or DNA
and produce measurable amounts of identical cDNA/
DNA amplicons by performing repetitive enzymatic
cycles. PCR primers (sequence specific oligos) make it
possible to pick out the specific sequence of a mRNA
target. Real-time PCR enables monitoring of accu-
mulated amplicons after each cycle due to labeling
of primers, probes or amplicons with fluorogenic
molecules (50;51). This approach has made it possible
to quantitate specific transcripts using either calibra-
tion curve methodology (52) or relative quantification
(ΔΔCt-method) (53).
Real-time PCR equipment offers the opportunity
to perform melting point analysis. Labelled, sequence
specific probes will have melting temperatures depend-
Foto: Henrik Alfthan. Island.
