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2. disease

PAXgene Blood RNA
®
The better the source, the more to explore.
Analyze RNA as if it
were still in the body
®
PAXgene Blood RNA System
®
Explore more at www.PreAnalytiX.com
One blood collection tube, many RNA purification options*
PreAnalytiX offers a complete preanalytical solution from blood collection to RNA purification, which
enables RNA-based research and molecular studies. The RNA purification step can now be performed
in a number of ways to meet your throughput and automation needs today and in the future.
• Manual or automated RNA purification from blood stabilized in PAXgene Blood RNA Tubes
• miRNA enrichment
Low Throughput
Manual
QIAcube®
Medium Throughput
QIAsymphony ®
High Throughput
96-well manual
* T he PAXgene Blood RNA System
is available for IVD use only when
composed of the PAXgene Blood
RNA Tube (762165) and the PAXgene
Blood RNA Kit (762164 or 762174)
02
BioRobot ®
An introduction to the PAXgene Blood RNA System
Key features and benefits of the PAXgene Blood RNA workflow
Detection and quantification of intracellular RNA transcripts.
Challenge
Solution
• R
NA transcript levels change dramatically, often within minutes of blood
collection, as a result of postphlebotomy gene induction, cell death, or enzymatic
RNA degradation. This can lead to artificial up or down regulation of gene
expression levels that are not representative of the real RNA transcript profile in
the body.
• Lack of standardized methods for RNA collection and purification from blood
increases variability in RNA quality and yield.
• Stabilize intracellular RNA at the point of blood collection.
• Integrate and standardize the workflow from blood collection to pure RNA.
The PAXgene Blood RNA System* is the first IVD marketed product for the collection, storage, and
transportation of blood and stabilization of intracellular RNA in a closed tube with subsequent isolation
and purification of intracellular RNA from whole blood for RT-PCR used in molecular diagnostic testing.
Performance characteristics for the PAXgene Blood RNA System have only been established with FOS
and IL1B gene transcripts. The user is responsible for establishing appropriate PAXgene Blood RNA
System performance characteristics for other target transcripts.
PAXgene Blood RNA workflow
Blood collection and
RNA stabilization
Transport
and storage
Purification
of total RNA
Molecular
RNA testing
PAXgene Blood RNA Tube:
PAXgene Blood RNA Kit:
• A single tube for blood collection, RNA stabilization,
• O
ptimized for purification of cellular RNA
specimen transport, and storage
• Prefilled with RNA stabilization reagent to provide
immediate RNA stabilization
• Blood cell lysis in tube simplifies RNA purification
• Consistent blood draw volume and blood-to-additive ratio
from stabilized blood
• H
igh-quality RNA
• Efficient and easy-to-follow manual or
automated protocol
• Multiple processing options available
* Only when composed of the PAXgene Blood RNA Tube (762165) in combination with the PAXgene Blood RNA Kit (762164 or 762174)
03
Transport and long-term storage of blood samples
Whether your blood specimens were in transport for a few days
or frozen for as long as 4 years,* the RNA test results will be unaffected.
Transport or store blood
specimens for
• 3 days at 18 – 25°C, or
• 5 days at 2 – 8°C
PAXgene
Blood RNA
Tubes
Purify RNA
manually or
automatically
High-quality
RNA ready
for analysis
Freeze tubes at –20 to –70°C
for as long as 4 years
RNA stability in blood samples stored at –20 and –70°C.
– 20° C
– 70° C
FOS / 18S rRNA RT-PCR
3
2
2
1
1
ΔΔCT
ΔΔCT
FOS / 18S rRNA RT-PCR
3
0
0
-- 1
-- 1
-- 2
-- 2
-- 3
-- 3
IL1B / 18S rRNA RT-PCR
3
2
2
1
1
ΔΔCT
ΔΔCT
IL1B / 18S rRNA RT-PCR
3
0
0
-- 1
-- 1
-- 2
-- 2
-- 3
-- 3
0
12
24
36
Blood sample storage (months)
48
0
12
24
36
48
Blood sample storage (months)
Blood was drawn into PAXgene Blood RNA Tubes from multiple donors and stored at –20 and –70°C. Total RNA was purified
manually with the PAXgene Blood RNA Kit. Relative transcript levels of FOS and IL1B were determined by real-time, duplex
RT-PCR, using 18S rRNA as an internal standard. The means are plotted, with standard deviations of all samples shown.
The dotted lines indicate the ±3x total precision of the assays (FOS/18S rRNA: 2.34 CT; IL1B/18S rRNA: 1.93 CT).
Transcripts stayed stable over time at both temperatures.
* Studies ongoing, see www.preanalytix.com
04
RNA stability in blood samples stored at room temperature (18–25°C) and
refrigerated (2–8°C).
FOS
18–25°C
EDTA
PAXGene Blood RNA
6
5
5
4
4
3
3
2
2
1
1
T
6
ΔΔC
ΔΔC
T
PAXGene Blood RNA
2–8°C
0
-1
0
-1
-2
-2
-3
-3
-4
-4
-5
-5
-6
EDTA
-6
day1
day2
day3
day1
day2
day3
day1
day2
day3
day4
day5
day1
day2
day3
day4
day5
day4
day5
IL1B
18–25°C
PAXGene Blood RNA
EDTA
6
5
5
4
4
3
3
2
2
1
1
T
6
0
ΔΔC
ΔΔC
T
PAXGene Blood RNA
2–8°C
-1
0
-1
-2
-2
-3
-3
-4
-4
-5
-5
-6
day1
day2
day3
day1
day2
day3
EDTA
-6
day1
day2
day3
day4
day5
day1
day2
day3
Replicate samples, from multiple donors, were collected in PAXgene Blood RNA Tubes and EDTA tubes and then stored as depicted above in a sub-set of matched samples. RNA was purified manually with the PAXgene Blood RNA Kit or a silica-membrane
based method at various time points as shown. Changes of the relative transcript levels of FOS and IL1B were determined by
real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The dotted lines indicate the ±3x total precision of the assays.
The small ΔΔCT values for the samples in PAXgene Blood RNA Tubes indicate that transcripts were not significantly changed. In
contrast, samples stored in EDTA tubes had a high transcript level variability independent of the day measured (for full details see
PAXgene Blood RNA Kit Handbook, www.preanalytix.com).
05
RNA purification (IVD)
• H
igh quality ready-to-use RNA (A260 /A280 ratio 1.8–2.2 for 95% of all samples)
• Consistent RNA yield (≥ 3 μg for > 95% of samples*)
• Genomic DNA contamination of less than 1% in ≥ 95% of all samples
• Reproducible and reliable quantitative RT-PCR results
• No significant RT-PCR inhibition when using up to 30% of the eluate
•
With automation:
• Less user interaction and hands-on time
• R
eduction of manual errors resulting in a higher degree of standardization
• Lower user-to-user variability
* for healthy donors with white blood cell (WBC) count in the range of 4.8–11.0 x 106 WBC/ml
06
Protocol performance
• The same kit can be used for manual or automated RNA purification on the QIAcube instrument.
• As part of the system, both protocols are available for in vitro diagnostic (IVD) use.*
RNA Yield
20
RNA Integrity
QIAcube protocol
Manual protocol
QIAcube protocol
Manual protocol
10
15
10
8.8
9.3
5
RNA Integrity Number [RIN]
RNA yield [μg/2.5 ml blood]
9
8
7
6
5
4
3
2
1
(A)
(B) 0
0
QIAcube Protocol
Manual Protocol
1
2
3
1
A
2
B
3
1
2
3
Mean
C
Combination of User (A,B,C) and Lot (1,2,3)
Blood samples were collected in PAXgene Blood RNA Tubes (576 tubes in total) and processed both manually and automatically
on multiple QIAcube instruments. To show reproducibility, multiple users (A, B, and C) used multiple kit lots (1, 2, and 3) with
both protocols.
(A) RNA yield was analyzed by UV spectroscopy. Median, lower/upper quartile, and min./max. RNA yields of all samples were
calculated and are shown for both protocols (n=288 per protocol).
(B) All RNA samples from one randomly chosen donor were selected for RNA integrity analysis. RIN values were calculated with
the Agilent® Bioanalyzer Nanochip. The means and standard deviations of RIN values from 36 samples per protocol were calculated and are shown in this figure (supporting data only).
There is no significant difference in the performance of the QIAcube and the manual procedure.
* The PAXgene Blood RNA System is available for IVD use only when composed of the PAXgene Blood RNA Tube (762165)
in combination with the PAXgene Blood RNA Kit (762164 or 762174).
07
RNA purification options (RUO)
• Fully integrated system allows manual and automated processing
• Choose the kits that are tailored to your specific needs
• Optimized miRNA protocol for co-purification of all RNAs
08
PAXgene Blood miRNA Kit (RUO)
• M
anual or automated (on QIAcube) purification
of total RNA including miRNA
• Low-throughput processing of 1 – 12 samples in parallel
miRNA Kit
QIAsymphony PAXgene Blood RNA Kit (RUO)
• Fully automated RNA purification
• Medium-throughput processing of 24 – 48 samples in parallel
QIAsymphony
PAXgene 96 Blood RNA Kit (RUO)
• Manual RNA purification
• High-throughput processing of 96 samples in parallel
96-well manual
PAXgene Blood RNA MDx Kit (RUO)
• F ully automated RNA purification
• High-throughput processing of 48 – 96 samples in parallel
BioRobot MDX or
BioRobot Universal System
RUO = for research use only, not for use in diagnostic procedures
09
miRNA purification
miRNA purification from samples collected in PAXgene Blood RNA Tubes
• Dedicated PAXgene Blood miRNA Kit co-purification of small RNAs (≥18 nt) with enrichment of miRNA
• Reliable quantification of miRNA in RT --PCR
• Reproducible results
Enrichment of small RNA species from PAXgene Blood miRNA Kit
RNA kit
Donor 1
miRNA kit
RNA kit
miRNA kit
Donor 4
Donor 4
[FU]
Small RNAs
60
[FU]
Small RNAs
40
40
30
20
20
Small
RNAs
RNA kit
10
0
0
30
35
40
Donor 2
miRNA kit
45
50
55
60
65
70
75
80 [s]
35
40
Donor 5
Small RNAs
100
50
55
60
65
70
75
80 [s]
Small RNAs
[FU]
60
80
40
60
40
Small
RNAs
45
Donor 5
[FU]
RNA kit
30
20
20
0
0
30
35
40
Donor 3
miRNA kit
45
50
55
60
65
70
75
80 [s]
30
40
Donor 6
50
60
70
80 [s]
Donor 6
Small RNAs
[FU]
Small RNAs
[FU]
200
100
150
100
50
Small
RNAs
(A)
50
0
0
30
35
40
45
50
55
60
65
70
75
80 [s]
30
40
50
60
(B)
High yields of small RNA species. Blood was collected in PAXgene Blood RNA Tubes and stored for 20 – 24 hours at
room temperature before freezing at -- 15 to -- 30°C. RNA was purified using the PAXgene Blood RNA Kit (RNA kit) or the
PAXgene Blood miRNA Kit (miRNA kit).
(A) Purified RNA was analyzed by agarose gel electrophoresis.
(B) Purified RNA was analyzed using the Agilent Small RNA LabChip. Small RNA species were significantly enriched
when using the PAXgene Blood miRNA Kit.
10
70
80 [s]
RT-PCR from PAXgene purified miRNA
26
Dedicated miRNA Kit
RNA Kit
24
Dedicated miRNA Kit
RNA Kit
1.000
22
20
CT value
18
1.000 E 1
16
14
12
1.000 E 2
(A)
0
5
10
15
20
CT value
25
30
35
40
10
(B)
Analysis of miRNA species using sensitive miScript assays. Blood was collected and RNA purified as described on page 10.
miRNAs were analyzed using the QIAGEN miScript PCR System, with the hsa-miR16 miScript Primer Assay.
The lower CT values for RNA purified using the PAXgene Blood miRNA Kit indicate higher amounts of miRNAs.
(A) Amplification plot of hsa-miR16 with PAXgene Blood miRNA Kit (miRNA kit) compared to PAXgene Blood RNA Kit (RNA Kit).
(B) Mean CT value of both kits.
11
PAXgene Blood RNA product selection guide
12
Multiple processing options
Depending on your requirements the PAXgene Blood RNA System can provide high-quality RNA and
miRNA from both manual and automated protocols.
Sample type
Blood
Blood collection tube
PAXgene Blood RNA Tube (IVD)
Nucleic acids
RNA
Procedure
Throughput
Manual
Low
Automated
High
Instrument
miRNA
Low
Medium
QIAcube
QIAsymphony
High
BioRobot
BioRobot
Universal
MDx
Manual
Automated
Low
Low
QIAcube
System
Kit
PAXgene
PAXgene 96
PAXgene
QIAsymphony
PAXgene
PAXgene
PAXgene
PAXgene
Blood RNA
Blood RNA
Blood RNA
PAXgene
Blood RNA
Blood RNA
Blood miRNA
Blood miRNA
Kit (IVD)
Kit (RUO)
Kit (IVD)
Blood RNA
MDx
MDx
Kit (RUO)
Kit (RUO)
Kit (RUO)
Kit (RUO)
Kit (RUO)
IVD = in vitro diagnostic; RUO = for research use only, not for use in diagnostic procedures
For further information on sample processing:
See the PreAnalytiX Web site www.preanalytix.com or contact QIAGEN technical services or your local QIAGEN distributor on
how to process your PAXgene Blood RNA Tubes.
13
The PAXgene Blood RNA Workflow
Collect blood according to instructions into a PAXgene
Blood RNA Tube.
Transport and store the filled PAXgene Blood RNA Tube as
described in the product circular. Incubate tubes for at least
2 hours at room temperature prior to processing.
Follow instructions in the kit handbook to pellet and wash
nucleic acids and transfer as instructed for processing.
Follow the manual or automated protocol of your chosen
PAXgene Blood RNA/miRNA Kit to purify high quality
RNA.
Purification is complete with a final elution and heat
denaturation of RNA.
0.6
0.5
0.4
OD
The PAXgene Blood RNA System results in high-quality
ready-to-use RNA in downstream applications as described in the intended use.
0.3
0.2
0.1
0
220
240
260
280
Wavelength (nm)
14
300
320
For up-to-date licensing information and productspecific disclaimers about PreAnalytiX products,
see the respective PreAnalytiX kit handbook or
user manual. PreAnalytiX kit handbooks and user
manuals are available at www.preanalytix.com or
can be requested from QIAGEN Technical Services or your local QIAGEN or BD distributor.
Trademarks:
PAXgene®, PreAnalytiX® (PreAnalytiX GmbH);
QIAGEN®, QIAcube®, QIAsymphony®, BioRobot®
(QIAGEN Group); Agilent® (Agilent Technologies,
Inc.).
1061043 01/2010 © 2010
PreAnalytiX, all rights reserved.
PreAnalytiX GmbH
Feldbachstraße
CH-8634 Hombrechtikon
www.preanalytix.com
®
Ordering Information
For more ordering information and online ordering go to:
www.preanalytix.com
Product
Contents
PAXgene Blood RNA Tubes (100)
100 PAXgene Blood RNA Tubes
PAXgene Blood RNA Kit (50)
50 PAXgene Spin Columns, 50 PAXgene Shredder
Spin Columns, Processing Tubes, RNase-Free DNase I,
RNase-Free Reagents and Buffers. To be used in conjunction with PAXgene Blood RNA Tubes.
QIAsymphony PAXgene Blood
For 96 preps: 2 Reagent Cartridges, Enzyme Racks,
RNA Kit (96)
Accessories, and RNase-Free Buffers. To be used in
Cat. no.
762165 * (WW)
762164
(North America)
762174 *
(Other Countries)
762535
conjunction with PAXgene Blood RNA Tubes.
PAXgene 96 Blood RNA Kit (4)
4 PAXgene 96 RNA Plates, 4 PAXgene 96 Filter Plates,
Buffers, Proteinase K, RNase-free DNase Sets, AirPore
762331
(Manual)
Tape Sheets, Collection Vessels. To be used in conjunction with PAXgene Blood RNA Tubes.
PAXgene Blood miRNA Kit (50)
For 50 RNA preps: PAXgene Spin Columns, PAXgene
763134
Shredder Spin Columns, Processing Tubes, Microcentrifuge Tubes, RNase-Free DNase, RNase-Free Reagents
and Buffers; to be used in conjunction with PAXgene
Blood RNA Tubes.
PAXgene Blood RNA MDx Kit (4)
For 4 x 96 RNA preps on the BioRobot MDx or Uni-
762431
versal System workstation: 4 PAXgene 96 RNA Plates,
4 PAXgene 96 Filter Plates, Buffers (wash buffers are
labeled with bar codes), Proteinase K, RNase-Free
DNase Set, Plasticware, Collection Vessels; to be used
in conjunction with PAXgene Blood RNA Tubes.
* Not available in all countries, please inquire
Discover more about
the PAXgene Blood RNA System at:
www.preanalytix.com
1061043 05/2010
®