PAXgene Blood RNA Kit

For isolation and purification of intracellular RNA from blood stabilized in PAXgene Blood RNA Tubes

Features

  • Compliance with EU IVD Directive 98/79/EC
  • Integrated system for collection, stabilization, and purification
  • RNA stabilization for up to 3 days at 18–25°C
  • Stabilization for at least 96 months at –20°C or –70°C
  • Standardized sample processing prior to analysis

 

*The QIAGEN QIAcube Connect MDx is not available in all countries. For further details please contact QIAGEN Technical Service.

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PAXgene Blood RNA Kit (50)

Cat. No. / ID: 762174

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
€626.00
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These products are not available in all countries; please inquire.
The PAXgene Blood RNA Kit is intended for in vitro diagnostic use.

✓ 24/7 automatic processing of online orders

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Product Details

The PAXgene Blood RNA system consists of PAXgene Blood RNA Tubes (available from BD, cat. no. 762165) for blood collection, stabilization, and transport, and the PAXgene Blood RNA Kit for silica-membrane-based RNA isolation and purification in a spin-column format. Purification can be carried out manually, using a microcentrifuge, or automated on the QIAcube Connect MDx. The CE-IVD-marked tubes and kit provide exact performance specifications, assuring highly reliable RNA purification.

Performance

PAXgene Blood RNA Tubes are intended for the collection of whole blood and stabilization of intracellular RNA for up to 3 days at 18–25°C (see figures "RNA stability at 18–25°C: FOS" and "RNA stability at 18–25°C: IL1B") or up to 5 days at 2–8°C (see figures " RNA stability at 2–8°C: FOS" and " RNA stability at 2–8°C: IL1B"). Currently available data shows stabilization of cellular RNA for at least 96 months at –20°C or –70°C.

Total RNA purified using the PAXgene Blood RNA System is highly pure, with A260/A280 values between 1.8 and 2.2 and ≤1.0% (w/w) genomic DNA. At least 95% of samples show no inhibition in RT-PCR, when using up to 30% of the eluate.
Average sample preparation time (based on data from 12 sample preps) is approximately 90 minutes, with only 40 minutes of hands-on time. RNA yields from 2.5 ml healthy human whole blood are ≥3 µg for ≥95% of the samples processed. Since yields are highly donor-dependent, individual yields may vary. For individual donors, the PAXgene Blood RNA System provides highly reproducible and repeatable yields (see figures " Reproducible and repeatable RNA purification" and " Repeatability and reproducibility of RNA yield") and reproducible and repeatable RT-PCR (see figures " Reproducibility between users" and " Reproducibility between kit lots", and table), making it highly robust for clinical diagnostic tests.

RNA yields from 2.5 ml healthy human whole blood are ≥3 μg for ≥95% of the samples processed. The figure " RNA yield and purity — automated processing" indicates the RNA yields from a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators. As pooled blood samples instead of individual PAXgene Blood RNA Tubes were used for these studies, the results do not reflect the RNA yield expected from single samples of individual blood draws. Since yields are highly donor-dependent, individual yields may vary.

At least 95% of samples show no inhibition in RT-PCR, when using up to 30% of the eluate. Using the automated protocol, cross contamination between samples is undetectable, as measured by quantitative, real-time RT-PCR of sequences of the betaglobin and FOS transcripts in RNA-negative samples (water) paired with RNA-positive samples (human whole blood) in the same run.

RNA purified with the PAXgene Blood RNA System and the automated protocol is highly pure, as shown by lack of RT-PCR inhibition (see above) and A260/A280 values between 1.8 and 2.2. Genomic DNA is present at ≤1% (w/w) in ≥95% of all samples, as measured by quantitative, real-time PCR of a sequence of the beta-actin gene. The figure " RNA yield and purity — automated processing" shows the A260/A280 values and relative genomic DNA of a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators. 

The automated protocol of RNA purification using the PAXgene Blood RNA System provides highly reproducible and repeatable RT-PCR results, as shown in the figure " Reproducibility between automated and manual protocols", making it highly robust for clinical diagnostic tests.

Summary of RT-PCR data
Test system  FOS/18S rRNA assay  IL1B/18S rRNA assay
Comparison of data  Mean  ± SD  Mean ± SD
   (ΔΔCT)  (ΔΔCT)  (ΔΔCT) (ΔΔCT)
 Reproducibility within each user and between all lots 
 All users, lot 1 – lot 1  0.00  0.00  0.00  0.00
 All users, lot 1 – lot 2  –0.03  0.48  –0.07  0.66
 All users, lot 1 – lot 3  –0.21  0.52  0.11  0.71
 Reproducibility within each lot and between all users 
 All lots, user A – user A  0.00  0.00  0.00  0.00
 All lots, user A – user B  –0.46  0.44  –0.06  0.69
 All lots, user A – user C  –0.31  0.60  –0.15  0.71
 

User: Technician, performed the study.
Lot: Number of kit lot used in this study.
SD: Standard deviation.
Mean ΔΔCT values (N = 120) and standard deviations are shown for the data presented in the figures " Reproducibility between users" and " Reproducibility between kit lots".

See figures

Principle

The PAXgene Blood RNA Kit is for the purification of total RNA from 2.5 ml human whole blood collected in a PAXgene Blood RNA Tube. The procedure is simple and can be performed using manual or automated procedures (see flowcharts " Manual PAXgene Blood RNA procedure" and " Automated PAXgene Blood RNA procedure"). 

PAXgene Blood RNA Tubes contain a proprietary reagent composition based on a patented RNA stabilization technology (US Patents 6,602,718 and 6,617,170). This reagent composition protects RNA molecules from degradation by RNases and minimizes ex vivo changes in gene expression. PAXgene Blood RNA Tubes are intended for the collection of whole blood and stabilization of intracellular RNA for up to 3 days at 18–25°C (see figures "  RNA stability at 18–25°C: FOS" and "  RNA stability at 18–25°C: IL1B") or up to 5 days at 2–8°C (see figures " RNA stability at 2–8°C: FOS" and " RNA stability at 2–8°C: IL1B"). Currently available data shows stabilization of cellular RNA for at least 96 months at –20°C or –70°C.

See figures

Procedure

The PAXgene Blood RNA procedure is simple and can be performed using manual or automated procedures (see flowcharts " Manual PAXgene Blood RNA procedure" and " Automated PAXgene Blood RNA procedure").

Manual PAXgene Blood RNA procedure

The resuspended pellet is incubated in optimized buffers together with proteinase K to bring about protein digestion. An additional centrifugation through the PAXgene Shredder spin column is carried out to homogenize the cell lysate and remove residual cell debris, and the supernatant of the flow-through fraction is transferred to a fresh microcentrifuge tube. Ethanol is added to adjust binding conditions, and the lysate is applied to a PAXgene RNA spin column. During a brief centrifugation, RNA is selectively bound to the PAXgene silica membrane as contaminants pass through. Remaining contaminants are removed in several efficient wash steps. Between the first and second wash steps, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA is eluted in elution buffer and heat-denatured.

Automated PAXgene Blood RNA procedure

Sample preparation, automated on the QIAcube Connect MDx, follows the same steps as the manual procedure, enabling you to continue using the PAXgene Blood RNA Kit for purification of high-quality RNA. 

The automated RNA purification protocol consists of 2 parts (or protocols), "PAXgene Blood RNA Part A" and "PAXgene Blood RNA Part B", with a brief manual intervention between the 2 parts.

The centrifuged, washed, and resuspended nucleic acid pellet is transferred from the PAXgene Blood RNA Tube into processing tubes, which are placed into the thermoshaker unit on the QIAcube Connect MDx worktable. The operator selects and starts the "PAXgene Blood RNA Part A" protocol from the menu. The QIAcube Connect MDx performs the steps of the protocol through to elution of RNA in elution buffer. The operator transfers the microcentrifuge tubes, containing the purified RNA, into the thermoshaker unit of the QIAcube Connect MDx. The operator selects and starts the "PAXgene Blood RNA Part B" protocol from the menu, and heat denaturation is performed by the QIAcube Connect MDx.

Average sample preparation time (based on data from 12 sample preps) is 125 minutes, with only approximately 20 minutes of hands-on time.

See figures

Applications

When the kit is used in conjunction with PAXgene Blood RNA Tubes, the system provides intracellular RNA from whole blood for RT-PCR used in molecular diagnostic testing.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsRT-PCR
FormatSpin column
Main sample typeWhole blood
Elution volume40 µl
Time per run90 min/12 samples
TechnologySilica technology
Yield>3 µg
Sample amount2.5 ml
ProcessingManual (centrifugation)
CE/FDA/IVD compatibleCE

Resources

Kit Handbooks (1)
Brochures & Guides (4)
For collection, transport, and storage of whole blood and stabilization and purification of intracellular RNA

 

Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Application Notes (2)
Typical total RNA yields from PAXgene Blood RNA Tubes processed with the PAXgene Blood RNA Kit
In situ stability of RNA in blood specimens stored for 11 years (132 months) at –20°C and –70°C in PAXgene Blood RNA Tubes

 

FAQ

Where can I find additional information for PreAnalytiX PAXgene products?
You can find additional information relating to the PreAnalytiX PAXgene products on the PreAnalytiX website .
FAQ ID - 3515
Can the PAXgene Blood RNA System be used with animal blood samples?

The PAXgene Blood RNA System was developed for intracellular RNA isolation from human blood samples. QIAGEN offers RNA stabilization and isolation systems for animal blood. Please contact QIAGEN technical service (www.qiagen.com) for more information.

FAQ ID - 3461
Can the PAXgene Blood RNA Tubes undergo freeze/thaw cycles?
PAXgene Blood RNA Tubes can go through up to two freeze/thaw cycles with no impact on yield or mRNA stability.
FAQ ID -2471
What RNA yields are expected with the PAXgene Blood RNA kit?

Typical yields of RNA isolated from 2.5 ml human whole blood are between 4 and 20 µg. However yield is highly donor-dependent, and in some cases higher or lower yields may be achieved. At least 95% of all samples with a WBC count between 4.8 and 11.0 x 106 cells/ml of blood will yield ≥3 µg of total RNA per tube. To prevent low yields due to underfilling of the tube, see the phlebotomy FAQ and the blood collection demonstration video.

FAQ ID - 3464
Why is a blood collection set required to be used with the PAXgene Blood RNA tube?

A blood collection set is required to be used with the PAXgene Blood RNA Tube to eliminate the possibility of patient contact with the reagent in the tube, due to backflow of blood, when used in accordance with the instructions for use.

FAQ ID - 3459
What is the integrity of RNA from PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue?
Like yield, RNA integrity depends on parameters, such as tissue type, period of cold ischemia, fixation time, processing protocol, age, and storage conditions of the PFPE block. For examples of the integrity of RNA in rat tissues under ideal workflow conditions, see Groelz et al., Exp Mol Pathol. 2013 Feb;94(1). For examples of the integrity of RNA from clinical samples, see Viertler et al., J Mol Diagn. 2012 Sep;14(5)
FAQ ID -3046
How long can the PAXgene Blood RNA tubes be kept frozen at –20°C or –70°C?
Currently we have data for storage at –20°C and –70°C for 96 months (In situ stability of RNA in blood specimens stored for 8 years (96 months) at –20°C and –70°C in PAXgene Blood RNA Tubes).  

Long term archiving studies are ongoing. See technical note: In Situ Stability of RNA in Blood Samples Stored at –20°C and –70°C in PAXgene Blood RNA Tubes.


 
FAQ ID - 3463
What are possible reasons for blood draws with lower than expected blood volume?

Possible reasons for blood draws with lower than expected blood volume include: 1) The phlebotomist has not waited enough time for the blood to stop flowing into the tube. 2) The tube has "pushed back" from the needle end attached to the holder and the tube is no longer drawing. 3) Discard tube was not used. For further information see the video Collecting Specimens In PAXgene Blood RNA Tubes and Instructions for Use.

FAQ ID - 3462
Is there any chance of the PAXgene Blood RNA tube reagent going back into the patient's arm?

If used according to the instructions for use, there is no possibility of backflow during phlebotomy, and thus no possibility of reagent going into the patient’s arm.

FAQ ID - 3460