Chroni
Biomedical
Page 9 of 10
Materials and Methods
Serum Samples. Blood from the tail
vein or artery was collected into 18 ml
plastic
tubes
equipped
with
a
coagulation accelerator and kept at room
temperature for 30 min to ensure proper
coagulation. Until further processing, the
tubes were stored at 2 - 8 °C for not
longer than 24 hours. Centrifugation was
done at 2 - 8 °C, 1000 x g for 15 min.
The serum supernatant was transferred
into 1.5 ml microcentrifuge cups in 0.5
ml aliquots and frozen immediately at
20 °C or -80 °C until use.
Sample Preparation. Frozen samples
were thawed at 4 °C in an ice-water bath
and 250 µl were transferred into a 1.5 ml
microcentrifuge tube. The tube was
centrifuged at 20,000 x g for 30 min at 4
°C in a Model 5214 bench top centrifuge
(Eppendorf, Hamburg, Germany). The
supernatant was carefully removed and
the pellet was used for further analyses.
Nucleic acid extraction. 20,000 x g
pellets were used with a standard silica
based
nucleic
acid
extraction
(NucleoMag; Macherey & Nagel, Düren,
Germany)
according
to
the
manufacturer's
instructions.
The
resulting nucleic acid solutions were
either used immediately or frozen at 80
°C until further use.
Diagnostic PCR. Three µL of the
extracted CNA were used in a PCR in a
total volume of 20 µL. Primers CHX-1F
and CHX-1R (Cat#: 42-4103 and 42-
91102, Chronix Biomedical GmbH,
Göttingen, Germany), were used at 1
µM each using a proofreading
polymerase system (Advantage-2 PCR
Kit,
BD-Clontech,
Heidelberg,
Germany). After 30 cycles of 95 °C for
30 sec, 55 °C for 45 sec, 68 °C for 1
min, a SybrGreenI (Cat#: S7563,
Molecular Probes, Eugene, OR, USA)
derived melting curve was recorded in a
MX4000 PCR system (Cat#: 401260,
Stratagene, La Jolla, CA, USA). The
area under the curve (AUC) of the
derived melting function d(F)/dT
between 87 °C and 90 °C was used for
analysis. Reactivity of each individual
sample was calculated on the basis of an
AUC above the detection limit, which is
defined as mean + 5 standard deviations
above baseline of non-template/normal
controls.
Statistical analysis. The proportion of
reactivity in the cohort groups and the
healthy control groups was calculated.
The statistical significance between the
cohorts and healthy controls was
estimated using the Chi-square test. For
total group comparisons, a calculation
with 9 degrees of freedom has been
done,
whereas
for
two-group
comparisons, the degrees of freedom
was lowered to 1.
References
1. Taback, B. and D. S. Hoon.
Circulating nucleic acids in plasma
and serum: past, present and future.
Curr Opin Mol Ther. 6(3):273-278,
2004.
2. Durie, B.G.M., H.B. Urnovitz and
W.H. Murphy. RNA in the Plasma
of Multiple Myeloma Patients:
Clinical Relevance and Molecular
Pathology. Acta Oncologica. 39:
789-796, 2000.
