Performance
The fully automated QIAsymphony PAXgene Blood RNA procedure provides RNA yields comparable to the manual PAXgene Blood RNA procedure. 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 with all PAXgene Blood RNA Kits. The automated QIAsymphony PAXgene Blood RNA procedure provides high-quality RNA from different donor samples with enrichment of small RNA species (Figure 1). Since yields are highly donor-dependent, individual yields may vary. The integrated DNase treatment removes trace genomic DNA and on-board measures of the QIAsymphony SP minimize cross-contamination (Figure 2).
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 proven magnetic particle technology, and when used in combination with PAXgene Blood RNA Tubes, the QIAsymphony PAXgene Blood RNA Kit provides a rapid, automated procedure to isolate intracellular RNA, including miRNA, of high quality and purity.
Procedure
The purification procedure starts with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube. After resuspension of the pellet, the tubes are transferred to the QIAsymphony SP. Loading of the reagent cartridge and plasticware is guided and checked by dedicated software. The QIAsymphony SP then carries out the fully automated RNA purification procedure. An integrated DNase treatment ensures that genomic DNA contamination is minimized (Figure 3). The robust and standardized procedure together with on-board measures of the QIAsymphony SP is designed to minimize the risk of sample-to-sample cross-contamination (Figure 2). After purification, a final heat treatment of the eluate enhances performance in downstream applications. Total processing time is about 2.5 hours for 24 samples, which includes a maximum of 25–30 minutes of hands-on time.
Applications
RNA purified using the QIAsymphony PAXgene Blood RNA Kit and the QIAsymphony SP 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
![Tip guards minimize the risk of sample-to-sample cross-contamination. [A] Pipetting channels. [B] Extractor unit.](/storage/images/Content/Product_pages/Blood/RNA_miRNA/On-board_measures_against_cross-contamination.jpg "Figure 2. On-board measures against cross-contamination.")
![[A] QIAsymphony SP instrument. [B] Schematic of the QIAsymphony SP principle.](/storage/images/Content/Product_pages/Blood/RNA_miRNA/QIAsymphony_PAXgene_Blood_RNA_procedure.jpg "Figure 3. QIAsymphony PAXgene Blood RNA procedure.")
