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PAXgene Blood RNA Kit (IVD)

For purification of intracellular RNA from whole blood to be used for in vitro diagnostic (IVD) tests

  • Coupled with the PAXgene Blood RNA Tube, constitutes the FDA-cleared, CE-marked PAXgene Blood RNA System
  • Compliance with EU IVD Directive 98/79/EC
  • Efficient purification of high-quality intracellular RNA
  • Standardized sample processing prior to analysis
  • Integrated DNase treatment for removal of gDNA
  • Purification can be automated on the QIAcube

Feature 

Specification

Format

Spin column

Technology

Silica membrane

Sample amount  

2.5 ml

Elution volume 

80 µl (2 x 40 µl)

Main sample type

Whole blood

Time per run
  • Manual: 90 min/12 samples
  • Automated: 125 min/12 samples

Typical RNA yield
≥3 µg/2.5 ml sample (≥95% of all samples processed)
RNA purity 
  • 1.8–2.2 (A260/A280)
  • ≤1 % (w/w) genomic DNA (≥95% of all samples processed)
Processing Manual: centrifugation
Automated: QIAcube

Intended Use

The PAXgene Blood RNA System consists of a blood collection tube (PAXgene Blood RNA Tube) and a nucleic acid purification kit (PAXgene Blood RNA Kit). It is intended for the collection, transport, and storage of blood and stabilization of intracellular RNA in a closed tube and subsequent isolation and purification of intracellular RNA from whole blood for RT-PCR used in molecular diagnostic testing.

Performance characteristics of the PAXgene Blood RNA System have 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.

Order information

Product

Catalog Nr.

Price

PAXgene Blood RNA Kit (50), CE

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.

762174
7450 CNY

Details

Performance

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 in ≥95% of all samples processed (Figure 1). At least 95% of samples show no inhibition in RT-PCR, when using up to 30% of the eluate. 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 (Figure 2  and Figure 3), making the System highly robust for clinical diagnostic tests.

The automated protocol of RNA purification using the PAXgene Blood RNA System provides pure RNA in high yield with reproducible and repeatable RT-PCR results, as shown in the figures (Figure 1 and Figure 4). Statistics of RT-PCR results are summarized in the table below. 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.

Table 1. Summary statistics of RT-PCR results using automated protocol

Test system 

 FOS/18S rRNA assay

 IL1B/18S rRNA assay

Comparison of data

Mean (ΔΔCT)

± SD (ΔΔCT)

Mean (ΔΔCT)

± SD (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, log 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.

Principle

Copy numbers of individual RNA species in blood can change significantly during storage or transport at ambient temperature, making reliable studies of gene expression difficult. Both stabilization of RNA molecules after collection and an efficient RNA purification protocol are needed to maximize insights into expression profiles of whole blood. Based on silica-membrane technology in spin column format, and when combined with PAXgene Blood RNA Tubes, the PAXgene Blood RNA Kit provides a rapid procedure and the chemistry to isolate RNA of high quality and purity.

Procedure

The PAXgene Blood RNA procedure is simple and can be performed using manual or automated procedures (Figure 5 and Figure 6).

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, resuspended, and 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, 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 precedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, resuspended, and transferred from the PAXgene Blood RNA Tube into processing tubes, which are placed into the thermoshaker unit on the QIAcube worktable. The operator selects and starts the "PAXgene Blood RNA Part A" protocol from the menu. The QIAcube 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. The operator selects and starts the "PAXgene Blood RNA Part B" protocol from the menu, and heat denaturation is performed by the QIAcube. 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.


The PAXgene Blood RNA Kit is for in vitro diagnostics (IVD).

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.Zoom image

Figure 1. RNA yield, purity and genomic DNA contamination – automated processing.

Blood samples from 48 different donors were collected in PAXgene Blood RNA Tubes ...
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.Zoom image

Figure 2. Repeatability and reproducibility of RNA yield.

Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes ...
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.Zoom image

Figure 3. Reproducible and repeatable RNA purification.

Quadruplicate blood samples from 14 donors were manually processed by each of 3 ...
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).Zoom image

Figure 4. Reproducibility between automated and manual protocols.

RNA was purified by 3 different operators (A, B, C) using 3 different lots (1, 2, ...
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.Zoom image

Figure 5. Manual PAXgene Blood RNA IVD procedure.

The PAXgene Blood RNA procedure begins with a centrifugation step to pellet ...
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.Zoom image

Figure 6. Automated PAXgene Blood RNA procedure on the QIAcube.

The PAXgene Blood RNA procedure begins with a centrifugation step to pellet ...

Resources

PAXgene Blood RNA Kit


1. What RNA yields are expected with this product?
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.

2. Can the PAXgene Blood RNA System be used to isolate viral RNA?
No. The PAXgene Blood RNA System has been optimized for cellular RNA only.

3. Can the PAXgene Blood RNA System be used to isolate DNA?
No. The PAXgene Blood RNA System has been optimized for cellular RNA only. PreAnalytiX offers the dedicated PAXgene Blood DNA Tube and a system (tube and kit) for collection and isolation of genomic DNA from human whole blood (see PAXgene Blood DNA System).

4. Can specific types of white blood cells be enriched from a PAXgene Blood RNA Tube prior to the RNA isolation?
No. The blood cells are lysed in the PAXgene Blood RNA Tube. There is no method available to enrich sub-populations of cells. BD offers the BD Vacutainer CPT Cell Preparation Tube for separation of mononuclear cells from whole blood. For more information, contact BD Global Technical Services (www.bd.com/vacutainer/).

5. Is RNA from red blood cells also isolated when RNA is extracted from a PAXgene Blood RNA Tube?
Yes. The PAXgene Blood RNA System purifies RNA from whole blood. Therefore, it will also purify RNA from reticulocytes and RBCs. Array analyses have shown higher globin mRNA levels for RNA isolated from whole blood compared to RNA isolated from PBMCs.

6. Can blood from leukemia patients with elevated numbers of white blood cells be processed?
We have not validated the system with blood specimens containing very high numbers of leukocytes. Blood samples highly exceeding the upper limit specified for WBCs (11.0 x 106 WBC/ml of blood) may lead to problems during the extraction procedure.

7. What is in the elution buffer?
The elution buffer is a proprietary low-salt buffer. In combination with the final heat denaturation step, it leads to optimal performance of the isolated RNA in RT-reactions. There is no interference with downstream applications reported to date.

8. Can water be used to elute the RNA?
No. The elution buffer supplied in the kit must be used.

9. As part of my RT procedure I routinely heat an aliquot of the eluate prior to the RT reaction. Do I still need to heat the whole eluate after the RNA preparation?
Yes. Heating temperature and incubation time will vary depending on different RT protocols. Therefore, the final heating step should be performed routinely at the end of the protocol.

10. Is a 260/230 nm absorbance ratio specified for RNA derived from the PAXgene Blood RNA System?
No. An  A260/A230 nm absorbance ratio is not specified for any PAXgene Blood RNA extraction kit. In contrast to a reduced A260/A280 ratio, which results from protein contamination of the eluted RNA and can lead to inhibitory effects in down stream applications, there is no negative impact of a reduced A260/A230 ratio on any down stream application reported to date and therefore it is not necessary to specify this.
In addition, because the elution buffer contains very low concentrations of salt that absorb at wavelengths between 220 and 230 nm, it is necessary to zero the photometer properly. Do this by preparing a solution for zeroing the photometer which contains a similar salt concentration as the sample to be measured. Add the same volume of Buffer BR5 (as the volume of RNA sample to be diluted) into a fresh volume of the buffer in which you will measure the absorption (we recommend 10 mM Tris Cl pH 7.5 buffer for this purpose).

11. Can the PAXgene RNA stabilization solution/blood mixture from the PAXgene Blood RNA Tube be disposed of by adding bleach?
Yes. You may dispose of the waste product from the PAXgene Blood RNA Tube with a 5% sodium hypochlorite bleach solution, in a 1:9 ratio (1 part bleach : 9 parts tube contents).

12. What is the proper disposal procedure for waste from the sample preparation?
Waste from the sample preparation, such as supernatants from centrifugation steps in the RNA purification process, is to be considered potentially infectious. Before disposal, the waste must be autoclaved or incinerated to destroy any infectious material. Disposal must follow official regulations.
 

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