PAXgene Blood RNA MDx Kit
For fully automated, high-throughput purification of cellular RNA from blood stabilized in PAXgene Blood RNA Tubes
The PAXgene Blood RNA MDx Kit is for fully automated, high-throughput purification of cellular RNA on the BioRobot MDx or BioRobot Universal System. (The PAXgene 96 Blood RNA Kit provides the same 96-well technology in a manual format.) Used with PAXgene Blood RNA Tubes (available from BD, cat. no. 762165) for sample collection and stabilization, the system provides a complete preanalytical solution for collection and stabilization through to purification of high-quality RNA to support high-throughput projects, such as clinical research studies.
For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
Performance
The automated PAXgene Blood RNA procedure provides high-quality RNA with high intra-donor repeatability (see figure "High-quality RNA from different donors"). Since yields are highly donor-dependent, individual yields may vary. An integrated DNase treatment ensures that genomic DNA contamination is minimized, typically to <0.1% (see figure "Very low levels of genomic DNA"). The fully automated PAXgene Blood RNA procedure provides RNA yields comparable to the manual PAXgene 96 Blood RNA procedure (typically ≥80%). Typical yields of RNA isolated from 2.5 ml healthy, human, whole blood (4.6 x 106 – 1.1 x 107 leukocytes/ml) are ≥3 µg for >95% of the samples processed. Purified RNA is ready to use in downstream applications, such as quantitative, real-time RT-PCR, with no RT-PCR inhibition detected (see figure "No RT-PCR inhibition detected"). Intersystem variation is minimal and well within the error range of typical real-time RT-PCR assays (see figure "High intersystem reproducibility"). The robust and standardized procedure is designed to minimize the risk of sample-to-sample cross-contamination. Total RNA was purified from 48 blood samples collected in PAXgene Blood RNA Tubes and 48 water samples. The samples were arranged in a checkerboard pattern on a 96-well block. After purification using the PAXgene Blood RNA MDx Kit on the BioRobot MDx workstation, all samples were analyzed by quantitative, real-time PCR using primers and probes specific for 18s rRNA for 40 PCR cycles. Threshold cycle values (CT) for the blood samples ranged from 27.3 to 32.7 while no RNA was detected in the water samples after 40 cycles.
Principle
Blood samples (2.5 ml) are collected directly into PAXgene Blood RNA Tubes, and RNA is isolated using the PAXgene Blood RNA MDx Kit. PAXgene Blood RNA Tubes contain a proprietary blend of reagents that bring about immediate stabilization of RNA. Blood samples can be stored in the PAXgene Blood RNA Tubes for up to 5 days at room temperature. RNA is then isolated by an automated procedure using silica membrane technology supplied in the PAXgene Blood RNA MDx Kit. The resulting RNA accurately represents the expression profile in vivo and is suitable for use in a range of downstream applications.
Procedure
The simple purification procedure begins with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube. Proteinase K and an optimized incubation buffer are added to digest proteins. The tubes are then transferred to the BioRobot MDx or BioRobot Universal System. After setup of buffers and plasticware, guided by the QIAsoft Operating System, the BioRobot workstation carries out the fully automated RNA purification procedure (see flowchart "Automated PAXgene RNA purification procedure"). Following purification, a final heat treatment of the eluate enhances performance in downstream applications. Total processing time is 3–4 hours, with a maximum of 25–30 minutes of hands-on time. Protocols are available for batches of 48 or 96 samples per run.
Applications
The purified RNA is ready for use in a wide range of downstream applications, including:
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