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Performance
The protocols of the PAXgene Blood miRNA Kit provide optimal binding conditions for several RNA species. The total RNA purified includes mRNA and RNA molecules smaller than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, tRNAs, and miRNAs (Figure 1). The miRNA species can be analyzed using the QIAGEN miScript PCR System (Figure 2) or other real-time RT-PCR systems.
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 silica-membrane technology in trusted spin column format, and when combined with PAXgene Blood RNA Tubes, the PAXgene Blood miRNA Kit provides the chemistry and rapid procedure to isolate high-quality total RNA and miRNA.
Procedure
Cell lysis and stabilization of intracellular RNA takes place upon collection of whole blood in PAXgene Blood RNA Tubes, effectively preventing changes to gene expression profiles by ex vivo gene regulation or RNA degradation. After removal of cellular debris, kit buffers provide optimal conditions for RNA molecules to bind to the silica membrane. Contaminants are washed away and trace DNA is removed by treatment with DNase I. Pure RNA, including miRNA, is then eluted in a low-salt elution buffer and denatured by heating (Figure 3).
Applications
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
![Blood was collected in PAXgene Blood RNA Tubes and stored for 20–24 hours at room temperature before freezing at –15 to –30°C. RNA was purified using the PAXgene Blood RNA Kit (RNA kit) or the PAXgene Blood miRNA Kit (miRNA kit). [A] Purified RNA was analyzed by agarose gel electrophoresis. [B] Purified RNA was analyzed using the Agilent Small RNA LabChip. Small RNA species were significantly enriched when using the PAXgene Blood miRNA Kit.](/storage/images/Content/Blood/RNA_miRNA/High_yield_of_small_RNA_species.jpg "Figure 1. High yields of small RNA species.")
