PAXgene Blood RNA Kit

Kit	Kit Content	Catalog No.
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	762164 (North America)
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	762174** (Other Countries)

Principle and procedure

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 “PAXgene Blood RNA Procedure”). In both protocols, purification begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and resuspended, followed by manual or automated RNA purification. In principle, both protocols follow the same protocol steps with the same kit components.
 
RNA stabilization in PAXgene Blood RNA Tubes

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 in Blood Samples at 18–25°C: FOS” and “RNA Stability in Blood Samples at 18–25°C: IL1B”) or up to 5 days at 2–8°C (see figures “RNA Stability in Blood Samples at 2–8°C: FOS” and “RNA Stability in Blood Samples at 2–8°C: IL1B”). Currently available data shows stabilization of cellular RNA for at least 50 months at –20°C or –70°C (for more information from ongoing studies evaluating stability for longer time periods, please contact QIAGEN technical services).

Highly reproducible and repeatable manual RNA purification

In detail, 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.
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 for Different Operators and PAXgene Blood RNA Kit Lots”) and reproducible and repeatable RT-PCR (see figures “Reproducibility of RT-PCR — Between Users” and “Reproducibility of RT-PCR — Between Kit Lots”, and table “Summary of RT-PCR Data”), making it highly robust for clinical diagnostic tests.

Automated RNA purification

Sample preparation using 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 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 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.

RNA yields from 2.5 ml healthy human whole blood are ≥3 μg for ≥95% of the samples processed. The figure “RNA Yield — 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 figures “RNA Purity (A₂₆₀/A₂₈₀ Values) — Automated Processing” and “RNA Purity (% Genomic DNA Contamination) — Automated Processing” show 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 of RT-PCR — Between Automated and Manual Protocols”, making it highly robust for clinical diagnostic tests.

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.

MSDS:

http://www1.qiagen.com/Support/Msds.aspx?catno=000000000000762174

*For in vitro diagnostic use. The PAXgene Blood RNA System consists of a blood collection tube (PAXgene Blood RNA Tube) and nucleic acid purification kit (PAXgene Blood RNA Kit). It is intended for the collection, storage, and transport 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.

**Not available in all countries, please inquire

Handbooks

PAXgene Blood RNA Kit (50) Handbook

Posters and Publications

In Situ Stability of RNA in Blood Samples Stored at –20°C and –70°C in PAXgene Blood RNA Tubes; Guenther et al., ISBER, 2009 Automated, Low-Throughput RNA Purification from Whole Blood Using the PAXgene Blood RNA System; Guenther et al., CHI Genomic Sample Prep, 2009 Maintaining the Stability and Integrity of RNA from Whole Blood Samples; Guenther and McCluskey, CLI, 2008 Automated, Low-Throughput RNA Purification from Whole Blood Using the PAXgene Blood RNA System; Guenther et al., DECHEMA, 2008 Development and Optimization of a Protocol for Automated RNA Purification Using the PAXgene Blood RNA System; Guenther et al., AACR, 2007 Performance Evaluation Study of the PAXgene Blood RNA System with Regulatory Compliance; Guenther et al., AMP, 2005

FAQs

PAXgene® Blood RNA CE/IVD Kit 1. What RNA yields are expected with this product? Answer Typical yields of RNA isolated from 2.5 ml human whole blood are between 4 and 20 µg. However yield is highly donor-dependant , 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/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? Answer 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? Answer No. The PAXgene Blood RNA System has been optimized for cellular RNA only. PreAnalytiX offers a dedicated system 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? Answer 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? Answer 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? Answer 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? Answer 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? Answer 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? Answer 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? Answer No. A 260/230 nm absorbance ratio is not specified for any PAXgene Blood RNA extraction kit. In contrast to a reduced 260/280 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 260/230 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? Answer 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. How are the supernatants resulting from the first two spin steps of the purification procedure disposed since they still may contain biologically hazardous material? Answer We recommend classifying the tube-processed supernatant as both a biological and a chemical waste. Supernatants can be decontaminated by autoclaving and disposed of as chemical waste. CAUTION: Do not add bleach or acidic solutions directly to the sample preparation waste. Disposal should be done in accordance with local, state and federal regulations.