PAXgene Blood RNA Kit
For isolation and purification of intracellular RNA from blood stabilized in PAXgene Blood RNA Tubes
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. The CE-IVD-marked tubes and kit provide exact performance specifications, assuring highly reliable RNA purification.
The PAXgene Blood RNA Kit is intended for in vitro diagnostic use.
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.
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.
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.
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, 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.
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.
fragment fix placeholder