paxgene blood rna - ibbl.lu€¦ · • manual or automated rna purification from blood stabilized...
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PAXgene® Blood RNA
The better the source, the more to explore.
PAXgene® Blood RNA SystemExplore more at www.PreAnalytiX.com
Analyze RNA as if it were still in the body
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
One blood collection tube, many RNA purification options*
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Manual
QIAcube®
QIAsymphony®
96-well manual
BioRobot®
Low ThroughpuT
MediuM ThroughpuT
high ThroughpuT
* The 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)
Key features and benefits of the PAXgene Blood RNA workflow
Detection and quantification of intracellular RNA transcripts.
• RNA 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
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One blood collection tube, many RNA purification options*
Challenge
Solution
PAXgene Blood RNA Tube:
• A single tube for blood collection, RNA stabilization,
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
PAXgene Blood RNA Kit:
• Optimized for purification of cellular RNA
from stabilized blood
• High-quality RNA
• Efficient and easy-to-follow manual or
automated protocol
• Multiple processing options available
Blood collection and RNA stabilization
Transport and storage
Purification of total RNA
Molecular RNA testing
An introduction to the PAXgene Blood RNA System
* Only when composed of the PAXgene Blood RNA Tube (762165) in combination with the PAXgene Blood RNA Kit (762164 or 762174)
Whether your blood specimens were in transport for a few daysor frozen for as long as 4 years,* the RNA test results will be unaffected.
RNA stability in blood samples stored at –20 and –70°C.
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Transport and long-term storage of blood samples
Transport or store blood specimens for• 3 days at 18 – 25°C, or• 5 days at 2 – 8°C
Purify RNA manually or automatically
Freeze tubes at –20 to –70°Cfor as long as 4 years
PAXgene Blood RNA Tubes
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
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High-quality RNA ready for analysis
FoS / 18S rrNA rT-pCr
iL1B / 18S rrNA rT-pCr
FoS / 18S rrNA rT-pCr
iL1B / 18S rrNA rT-pCr
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RNA stability in blood samples stored at room temperature (18–25°C) and refrigerated (2–8°C).
Replicate samples, from multiple donors, were collected in PAXgene Blood RNA Tubes and EDTA tubes and then stored as depict-
ed 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).
FoS
pAXgene Blood rNA edTA
2–8°C18–25°C
pAXgene Blood rNA edTA
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pAXgene Blood rNA edTA
2–8°C18–25°C
pAXgene Blood rNA edTA
iL1B
• High 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 • Reduction of manual errors resulting in a higher degree of standardization • Lower user-to-user variability
RNA purification (IVD)
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* for healthy donors with white blood cell (WBC) count in the range of 4.8–11.0 x 106 WBC/ml
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.*
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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 calcu-
lated and are shown in this figure (supporting data only).
There is no significant difference in the performance of the QIAcube and the manual procedure.
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QIAcube Protocol Manual Protocol
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* 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).
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
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PAXgene Blood miRNA Kit (RUO)
• Manual or automated (on QIAcube) purification of total RNA including miRNA
• Low-throughput processing of 1 – 12 samples in parallel
QIAsymphony PAXgene Blood RNA Kit (RUO)
• Fully automated RNA purification• Medium-throughput processing of 24 – 48 samples in parallel
PAXgene 96 Blood RNA Kit (RUO)
• Manual RNA purification• High-throughput processing of 96 samples in parallel
PAXgene Blood RNA MDx Kit (RUO)
• Fully automated RNA purification • High-throughput processing of 48 – 96 samples in parallel
miRNA Kit
QIAsymphony
96-well manual
BioRobot MDX or BioRobot Universal System
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RUO = for research use only, not for use in diagnostic procedures
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
Donor 5
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.
miRNA purification
(B)
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(A)
Donor 1RNA kit miRNA kit
SmallRNAs
Donor 3RNA kit miRNA kit
SmallRNAs
Donor 2RNA kit miRNA kit
SmallRNAs
Donor 4 Donor 4
RNA kit miRNA kit
Donor 5
Donor 6 Donor 6
Small RNAs
Small RNAs
Small RNAsSmall RNAs
Small RNAs
Small RNAs
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RT-PCR from PAXgene purified miRNA
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.
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Dedicated miRNA Kit
RNA Kit
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PAXgene Blood RNA product selection guide
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Throughput
Instrument
Kit PAXgene
Blood RNA
Kit (IVD)
QIAsymphony
QIAsymphony
PAXgene
Blood RNA
Kit (RUO)
BioRobot
Universal
System
PAXgene
Blood RNA
MDx
Kit (RUO)
BioRobot
MDx
PAXgene
Blood RNA
MDx
Kit (RUO)
PAXgene
Blood miRNA
Kit (RUO)
QIAcube
PAXgene
Blood miRNA
Kit (RUO)
PAXgene 96
Blood RNA
Kit (RUO)
Procedure Manual Automated Manual Automated
Nucleic acids miRNA
Blood collection tube
Sample type Blood
PAXgene Blood RNA Tube (IVD)
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.
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.
QIAcube
PAXgene
Blood RNA
Kit (IVD)
Low High Low Medium High Low Low
RNA
Collect blood according to instructions into a pAXgene Blood rNA Tube.
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The PAXgene Blood RNA Workflow
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.
The pAXgene Blood rNA System results in high-quality ready-to-use rNA in downstream applications as descri-bed in the intended use.
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Wavelength (nm)
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0220 240 260 280 300 320
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For up-to-date licensing information and product-
specific 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 Servi-
ces 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
®
PAXgene Blood RNA Tubes (100)
PAXgene Blood RNA Kit (50)
QIAsymphony PAXgene Blood RNA Kit (96)
PAXgene 96 Blood RNA Kit (4)
PAXgene Blood miRNA Kit (50)
PAXgene Blood RNA MDx Kit (4)
1061043 05/2010
Ordering Information
Product Contents Cat. no.
100 PAXgene Blood RNA Tubes
50 PAXgene Spin Columns, 50 PAXgene Shredder Spin Columns, Processing Tubes, RNase-Free DNase I, RNase-Free Reagents and Buffers. To be used in con-junction with PAXgene Blood RNA Tubes.
For 96 preps: 2 Reagent Cartridges, Enzyme Racks, Accessories, and RNase-Free Buffers. To be used in conjunction with PAXgene Blood RNA Tubes.
4 PAXgene 96 RNA Plates, 4 PAXgene 96 Filter Plates, Buffers, Proteinase K, RNase-free DNase Sets, AirPore Tape Sheets, Collection Vessels. To be used in conjunc-tion with PAXgene Blood RNA Tubes.
For 50 RNA preps: PAXgene Spin Columns, PAXgene Shredder Spin Columns, Processing Tubes, Microcentri-fuge Tubes, RNase-Free DNase, RNase-Free Reagents and Buffers; to be used in conjunction with PAXgene Blood RNA Tubes.
For 4 x 96 RNA preps on the BioRobot MDx or Uni-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.
762165 * (WW)
762164(North America)
762174 * (Other Countries)
762535
762331 (Manual)
763134
762431
discover more about the pAXgene Blood rNA System at: www.preanalytix.com
For more ordering information and online ordering go to:www.preanalytix.com
* Not available in all countries, please inquire
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