1. No category

Highest
g
sample
p q
quality
y for molecular analysis
y
through
g ambient stabilization technologies.
technologies
g
I
Improved
d genome, transcriptome
t
i t
and
d proteome
t
analysis
l i from
f
saliva,
li
bl d and
blood
d tissue
ti
samples.
l
Ste e Wilkinson,
St
Steven
Wilki so , Vasco
Vasco Liberal,
Lib
be al, Scott
S tt Whitney,
Whitt ey, Senait
Se
S aitt Ghirmai,
G
Ghi ai, Angela
A gela Stassinopoulos,
Stass
St
i opoulos, Winnie
Wi ie Huang,
Hua g, Lutfunnessa
Luttfu essa Shireen,
S
Shi ee ,
R lf M
Rolf
Muller
ll and
d Judy-Muller
J dy M ll Cohn
C h
Biomatrica Inc.,
Biomatrica,
Inc 5627 Oberlin Drive,
Drive Suite 120,
120 San Diego,
Diego
g CA 92121
Abstract
Biospecimen stabilization during collection and transport is of critical
importance
p
in the fields of biomedical research and molecular
diagnostics The emergence of personalized medicine has placed
diagnostics.
an even greater emphasis on the significance of protecting the
q alit and reliability
quality
reliabilit of clinical samples.
samples Current
C rrent methods for blood
and saliva collection depend heavily on cold-chain logistics. The
reliance on cold and ultra-cold based sample
p p
preservation is fraught
g
with potential complications to specimen integrity that can result in
inconsistent result generation,
generation impacting the discovery of suitable
and reliable biomarkers and their transition to clinical applications.
applications
Ch i l stabilization
Chemical
t bili ti
off biomolecules
bi
l
l
att ambient
bi t temperatures
t
t
i
is
an effective alternative to low temperature
p
stabilization methods in
preserving sample quality and integrity.
In this report we describe the development of chemical stabilization
t h l i
technologies
th t address
that
dd
th problem
the
bl
off sampling
li
i d
induced
d
inconsistencies in human blood, plasma and saliva. The
formulations described p
preserve nucleic acids in these
biospecimens even under extreme environmental shipping
conditions and remove the need for cold-chain logistics during
sample collection,
collection transport or storage.
storage We show that the genomic
DNA in
i blood
bl d collected
ll t d in
i DNAgard
DNA d Blood
Bl d Tubes
T b is
i stabilized
t bili d during
d i
extreme temperature
p
fluctuations ranging
g g from -20°C to +45°C and
during prolonged storage at room temperature. The analogous DNA
stabilization technology for saliva specimens,
specimens DNAgard Saliva,
Saliva
preserves sample integrity for over 12 months at ambient
t
temperatures.
t
W also
We
l
compare freezer
f
storage
t
with
ith the
th room
temperature stabilization formulation RNAgard Blood and other
technologies
g
for ambient temperature
p
blood RNA and p
protein
stabilization
stabilization.
Vacutainers
V
t i
and
d in
i a competitor’s
tit ’ blood
bl d collection
ll ti
t b
tubes
were
subjected to an 8 day shipping simulation (A). Blood samples collected
in K2-EDTA Vacutainers were stored at -20°C as controls. DNA was
purified and analyzed by long
long-range
range PCR amplification of a 22 kbp
region of the tPa gene (B).
(B) Equal input DNA was used for each
amplification reaction.
reaction L = lambda DNA-HindIII digest (New England
Biolabs) Also shown are representative (-)
Biolabs).
( ) Taq and (-)
( ) DNA template
control
t l samples.
l
A.
B.
Call Rates
Call Rates
Donor
DNAgard
Blood
Frozen
1
0 9979994
0.9979994
0 9975027
0.9975027
2
0 9927651
0.9927651
0 9931350
0.9931350
3
0.9970026
0.9984962
4
0 9982392
0.9982392
0 9985784
0.9985784
DGS
A.
NP
-80°C
Results I – genomic DNA stabilization in blood
A
A.
Temperature Cycle
Temperature
Time
Room temperature
p
2 days
y
37°C
2 days
2 days
Room temperature
p
2.5 days
y
‐20°C
20°C
1 day
1 day
45°C
45
C
1 day
day
B
B.
L
K2-EDTA
32.21
32 21
30.10
30 10
A 260/280
1 88
1.88
1 91
1.91
Figure 3: Preservation of DNA in saliva for over a year at 50
50°C
C
versus saliva stored in -80°C
80°C freezer.
freezer Fresh saliva (200 µL) was
mixed
i d with
ith DNAgard
DNA d Saliva
S li (DGS) (150 µL)
L) and
d stored
t d att 50°C for
f 14
months. Aliquots of saliva were stored at 50°C and -80°C as negative
((NP)) and p
positive controls,, respectively.
p
y Purified DNA from was
analyzed by gel electrophoresis (A). DNA quantification demonstrated
DNA recovery equivalent to that from frozen controls (B).
(B)
K2-EDTA,
EDTA, -20°C
20 C
A.
IL-12p70
p
B.
Figure 6. Microarray analysis in blood stored in RNAgard
Blood Tubes.
Tubes Human blood from 2 healthy donors was
collected in RNAgard tubes or competitor
competitor’ss RNA preservation
t b and
tubes
d RNA isolated
i l t d on collection
ll ti day
d and
d after
ft 3 or 14 days
d
t
t
t
t t d in
i materials
t i l and
d
off room temperature
storage,
as stated
methods. A-D: Representative analysis, for Donor 2, of gene
expression
p
profile of ~34000 g
p
genes using
g the Illumina Human
HT12 Bead Array, for RNA extracted from blood collected in
RNAgard Blood or competitor
competitor’ss Tubes after 3 days (A,C)
(A C) or 14
days (B,D)
(B D) of room temperature storage,
storage relative to RNA
extracted from freshly collected blood,
blood using RNA isolation
methods
th d recommended
d d for
f each
h product.
d t E:
E Table
T bl summarizing
i i
the microarray results. Displayed are the number of genes with
>2-fold change
g in expression
p
levels relative to samples
p
processed in the day of blood collection.
600
1000
900
500
800
700
600
500
400
300
200
0
-80
80 -20
20 4
NP 5 6 7 8 9 10 11 12
C C C
pg/ml 548 577 612 344 408 536 526 522 538 567 671 466
DNAgard Blood protects DNA integrity in whole blood stored
at ambient temperature:
400
300

DNAgard Blood protects DNA in blood samples subjected
to harsh shipping
pp g conditions ((figure
g
1).
)

Blood storage in DNAgard Blood at room temperature is as
effective as freezer storage for genotyping (figure 2).
2)
200
100
100
Summary
IL-1β
β
0
-80 -20 4
NP 5 6 7 8 9 10 11 12
C C C
pg/ml 511 450 299 23. 165 443 480 438 370 483 370 433
pg
Figure 4.
4 Protein stabilization in human plasma at elevated
temperatures Human IL-12p70
temperatures.
IL 12p70 (A) and IL-1β
IL 1β (B) were spiked into
plasma
l
and
d protein
t i levels
l
l measured
d by
b flow
fl
cytometry
t
t after
ft 4 days
d
off
incubation at 37°C. Test formulations were comprised of stabilization
compounds
p
titrated into a “base formulation”, lane 5 in each figure.
g
Control
Co
t o sa
samples
p es without
t out added formulation
o u at o were
e e incubated
cubated at -80°C,
80 C,
4°C
4
C or at the test temperature (unprotected; NP).
NP)
DNAgard
Blood
DNAgard
DNA
d Saliva
S li
protects
t t DNA integrity
i t
it in
i saliva
li
samples
l
stored at ambient temperature:
p

DNAgard Saliva preserves genomic DNA integrity in saliva
samples for over one year at ambient temperature (more
th six
than
i years based
b
d on accelerated
l
t d aging
i
(fi
(figure
3)
3).

DNAgard
g
Saliva p
provides high
g DNA recovery
y and better
quality compared to freezer control samples.
Protein stabilization in plasma at room temperature

Small
S
ll molecule
l
l stabilizers
t bili
protect
t t labile
l bil proteins
t i in
i h
human
plasma figure
g
4).
)
RNAgard Blood protects RNA in Blood in blood stored at
room temperature:
Comp.
Figure
g
1. Genomic DNA stability
y in whole blood samples
p
subjected to extreme temperature fluctuations. Blood from 5
donors collected in Vacuette DNAgard Blood Tubes,
Tubes K2-EDTA
E: Number of genes > 2-fold expression variation
Results III – Protein stabilization in plasma
Results IV – RNA stabilization in blood
L
D: Competitor, Dayy 14
-80 °C
ed
DNA y
yield
(µg/mL)
[Prottein]] pg//ml
RNA stabilization in whole blood:
Human whole blood was collected in RNAgard Blood tubes,
tubes and stored at
room temperature.
temperature Blood was also collected in a competitor
competitor’ss tubes and
stored at room temperature.
temperature RNA was isolated using Biomatrica
Biomatrica’ss BioMaxi
system (Figure 5,
5 6) or by a competitor
competitor’ss system.
system For RT-qPCR,
RT qPCR fold-change
fold change
in gene expression was calculated relative to expression at the time of
collection ((“time
time 0
0”,)) using the ∆∆Cq method of relative quantification.
quantification Gene
expression analysis was performed with the Human HT-12
HT 12 array (Illumina).
(Illumina)
C Competitor, Dayy 3
C:
B.
DGS
[Prottein]] pg//ml
Protein stabilization in plasma:
Tumor necrosis factor (TNF; Becton Dickinson) was spiked into aliquots of
human plasma at a final concentration of 1.25
1 25 ng/ ml.
ml Test samples were
mi ed with
mixed
ith Biomatrica formulations
form lations to give
gi e a final concentration of 0.5x
05
plasma.
l
C t l samples
Control
l
without
ith t added
dd d formulation
f
l ti
were stored
t d att the
th
t
temperatures
t
i di t d TNF stability
indicated.
t bilit was measured
d by
b flow
fl
cytometry
t
t using
i
th BD CBA TNF Flex
the
Fl Set
S t with
ith an Accuri
A
i C6 flow
fl
cytometer.
t
t
B: RNAgard
RNA
d Blood,
Bl d Day
D 14
Results II – DNA stabilization in saliva
Materials and Methods
DNA Stabilization in saliva:
DNA was isolated using the QIAamp DNA mini kit (QIAGEN). DNA integrity
was analyzed on a 0.8% agarose gel and 10% of the eluted DNA was
loaded to the gel. DNA yield was determined using the Quant-iTTM
PicoGreen dsDNA Assay
y Kit ((Invitrogen).
g )
A: RNAgard
RNA
d Blood,
Bl d Day
D 3
DNAgard
Blood K2-EDTA
Figure 2. Protection of DNA integrity in whole blood at ambient
temperature.
p
((A)) Blood from four separate
p
donors was collected in
Vacuette DNAgard Blood tubes and stored at ambient temperature
and collected in K2-EDTA
EDTA tubes and stored frozen at -20°C
20 C. After 5.5
55
months of storage,
storage DNA was purified and genotyped on the Human
CytoSNP 12 SNP Genotyping Array (Illumina).
CytoSNP-12
(Illumina) (B) The gDNA from
bl d stored
blood
t d in
i Vacuette
V
tt DNAgard
DNA d Blood
Bl d tubes
t b
f
for
9 months
th att
ambient temperature was compared by agarose gel electrophoresis to
the g
gDNA isolated from blood stored in K2-EDTA tubes under the same
conditions or frozen at -20°C ((samples
p
were analyzed
y
in triplicate).
p
)
The results
res lts presented demonstrate that transcriptome and protein
b
d analysis
l i is
i vastly
l improved
i
d through
h
h ambient
bi
bi
bili i
based
biostabilization
g
technologies.
DNA stabilization in whole blood:
DNA from human whole blood stored ± DNAgard
g
Blood was isolated using
g
QIAamp Blood Maxi kits (QIAGEN) or by the competitor
competitor’ss specified method.
Long range PCR analysis was conducted with 250 ng of input DNA and
primers to amplify a 22 kbp amplicon of the tPa gene. Genotyping was
performed on the Human CytoSNP
CytoSNP-12
12 SNP Genotyping Array (Illumina).
(Illumina)
-20°C
-20
C
room temperature
t
t
f 14 days.
for
d
RNA was purified
ifi d from
f
RGB samples
l
using
i
Bi
Biomatrica’s
t i ’ BioMaxi
Bi M i RNA Kit and
d from
f
th competitor’s
the
tit ’ tubes
t b
with their recommended kit. 200 ng of purified RNA from each sample
was analyzed
y
byy agarose
g
gel electrophoresis.
g
p
Figure 5. Room temperature stabilization of RNA in whole blood.
Blood from four separate human donors was collected in RNAgard
Blood Tubes (RGB) or in a competitor
competitor’ss blood tube and stored at

The gene expression profile in blood stored for 3 and 7
days
y at room temperature
p
in Biomatrica RNAgard
g
Blood is
similar to fresh blood, outperforming a leading competitor
(figure 6 A,
A B).
B)

Gene expression remains unaltered in blood samples
stored
t
d for
f 14 days
d
att room ttemperature
t
i RNAgard
in
RNA
d Blood
Bl d
(f
(figure
6 D, E).
)