text.skipToContent text.skipToNavigation

PAXgene Blood RNA Kit

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

  • 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.

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.

Buy Products

Cat No./ID: 762174
PAXgene Blood RNA Kit (50)
Inquire
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
These products are not available in all countries; please inquire.

The PAXgene Blood RNA Kit is intended for in vitro diagnostic use.


Product Details

9
In vitro diagnostic medical device.
 
9
CE-IVD-marked.
 
9
RNA yield and purity — automated processing.
Blood samples from 48 different donors were collected in PAXgene Blood RNA Tubes (6 tubes per donor, 288 tubes in total). The contents of the tubes from 6 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 6-donor-pool were processed by 3 different operators (A, B, C). Each operator used 3 different lots (1, 2, 3) of the PAXgene Blood RNA Kit for automated extraction and processed quadruplicate samples from each of the 8 donor pools. RNA yields, A260/A280 values, and genomic DNA amounts (w/w) of all individual samples are shown for every operator–lot combination.
9
Manual PAXgene Blood RNA procedure.
The PAXgene Blood RNA procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and resuspended, followed by manual RNA purification.
9
Reproducibility between users.
Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes (12 tubes per donor, 360 tubes in total). The contents of the tubes from 3 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 3-donor-pool were manually processed by 3 different operators. Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools. Relative transcript levels of  [A]  FOS and  [B]  IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. The values for all samples are plotted, relative to the values for user 1 (10 donor pools x 3 kit lots x 4 replicates = 120 data sets for each gene), with means (red lines) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS, 2.34 CT; IL1B, 1.93 CT).
9
Reproducibility between kit lots.
Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes (12 tubes per donor, 360 tubes in total). The contents of the tubes from 3 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 3-donor-pool were manually processed by 3 different operators. Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools. Relative transcript levels of  [A]  FOS and  [B]  IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. The values for all samples are plotted, relative to the values for kit lot 1 (10 donor pools x 3 users x 4 replicates = 120 data sets for each gene), with means (red lines) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS, 2.34 CT; IL1B, 1.93 CT).
9
Automated PAXgene Blood RNA procedure.
The PAXgene Blood RNA procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and resuspended, followed by automated RNA purification.
9
RNA stability at 2–8°C: IL1B.
Blood was drawn from 10 donors, with duplicate samples, and stored at 2–8°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane-based RNA cleanup. Relative transcript levels of IL1B were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (1.93 CT).
9
RNA stability at 18–25°C: IL1B.
Blood was drawn from 10 donors, with duplicate samples, and stored at 18–25°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane–based RNA cleanup. Relative transcript levels of IL1B were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (1.93 CT).
9
Reproducibility between automated and manual protocols.
RNA was purified by 3 different operators (A, B, C) using 3 different lots (1, 2, 3) of the PAXgene Blood RNA Kit using the automated protocol in the experiment described in the figure "RNA yield and purity — automated processing". In parallel, RNA was purified from the corresponding replicate tubes using the manual protocol. Relative transcript levels of  [A]  FOS and  [B]  IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. Possible differences of transcript levels between RNA prepared from paired blood samples using both extraction protocols (automated and manual protocol) were calculated by the ΔΔCT method. Individual ΔΔCT values for all sample pairs (4 replicates x 8 donor pools x 3 kit lots x 3 operators = 288 pairs for each gene) are plotted as single dots with means (larger dots) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS: 2.34 CT; IL1B, 1.93 CT).
9
Repeatability and reproducibility of RNA yield.
Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes (12 tubes per donor, 360 tubes in total). The contents of the tubes from 3 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 3-donor-pool were manually processed by 3 different operators. Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools.  [A]  RNA yield and standard deviation for every operator–lot combination. Quadruplicate blood samples from 10 donor pools were processed by 3 different operators (A, B, C) with each of 3 kit lots (1, 2, 3). The mean yields (columns) and standard deviations (error bars) per quadruplicate sample from the same donor pool for different operator and different kit lot are presented.  [B]  CV of RNA yield per donor pool for all operator–lot combinations (A, B, C; 1, 2, 3) as calculated from the mean yield and standard deviation of the yield shown in part A.
9
RNA stability at 18–25°C: FOS.
Blood was drawn from 10 donors, with duplicate samples, and stored at 18–25°C for the indicated number of days, followed by total RNA purification.  [A]  Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit.  [B]  Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane–based RNA cleanup. Relative transcript levels of FOS were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (2.34 CT).
9
RNA stability at 2–8°C: FOS.
Blood was drawn from 10 donors, with duplicate samples, and stored at 2–8°C for the indicated number of days, followed by total RNA purification.  [A]  Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit.  [B]  Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane-based RNA cleanup. Relative transcript levels of FOS were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (2.34 CT).
9
Reproducible and repeatable RNA purification.
Quadruplicate blood samples from 14 donors were manually processed by each of 3 technicians (A, B, C). Three sets of equipment were used, and all samples prepared by a single technician were processed using the same equipment.  [A]  Means and standard deviations of RNA yield per replicate samples from the same donors and different technicians are shown.  [B]  Twelve replicate blood samples from each of 14 donors were processed by the 3 different technicians. Means and standard deviations of RNA yield per samples from the same donors and all technicians are presented. For all RNA samples, A260/A280 ratios ranged from 1.8 to 2.2.
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".

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.

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.

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.

Specifications

Features
Specifications
Applications RT-PCR
CE/FDA/IVD compatible CE
Elution volume 40 µl
Format Spin column
Main sample type Whole blood
Processing Manual (centrifugation)
Sample amount 2.5 ml
Technology Silica technology
Time per run 90 min/12 samples
Yield >3 µg

Product Resources

You are not authorized to download the resource

Application Notes (2)
PAXgene Blood RNA System Stability Technical Note
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

 


 Show details
PAXgene Blood RNA System Yield Technical Note
Typical total RNA yields from PAXgene Blood RNA Tubes processed with the PAXgene Blood RNA Kit
Show details
fragment fix placeholder
Privacy Policy Sitemap Trademarks & Disclaimers Accessibility