The automated PAXgene Blood RNA MDx procedure provides high-quality RNA (Figure 1). An integrated DNase treatment ensures that genomic DNA contamination is minimized, typically to <0.1% (Figure 2). The fully automated PAXgene Blood RNA MDx 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. Since yields are highly donor-dependent, individual yields may vary. Purified RNA exhibits no detectable RT-PCR inhibition (Figure 3) and intersystem variation is minimal and well within the error range of typical real-time RT-PCR assays (Figure 4).
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.
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 proven silica-membrane technology in a high-throughput, 96-well plate format, and when combined with PAXgene Blood RNA Tubes, the PAXgene 96 Blood RNA MDx Kit provides a rapid, automated procedure to isolate RNA of high quality and purity.
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 (Figure 5). 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.
The purified RNA is ready for use in a wide range of downstream applications, including:
- cDNA synthesis
- Gene expression arrays
- End-point RT-PCR
- Quantitative RT-PCR, including TaqMan technology
- Next-generation sequencing