PAXgene Blood miRNA Kit, 50 preps

For 50 RNA preps: PAXgene Spin Columns, PAXgene Shredder Spin Columns, Processing Tubes, Microcentrifuge Tubes, RNase-Free DNase, RNase-Free Reagents and Buffers; to be used in conjunction with PAXgene Blood RNA Tubes

Features

• Effective isolation of intracellular RNA, including miRNA
• Standardized sample processing prior to analysis
• Integrated DNase treatment for removal of gDNA
• Isolation can be automated on QIAcube instruments

Product Details

The PAXgene Blood miRNA System consists of the PAXgene Blood miRNA Kit for purification of total RNA, including RNA longer than approximately 18 nucleotides, and PAXgene Blood RNA Tubes (available from BD, cat. no. 762165) for collection, stabilization, and transport of blood. Purification is carried out using silica-based RNA purification technology in a spin-column format. Purification can be carried out manually, using a microcentrifuge, or automated on the QIAcube Connect. Used with the tubes, the kit provides a complete preanalytical solution for collection and stabilization through the purification of high-quality total RNA, including miRNA, for molecular analysis.

Performance

The protocols of the PAXgene Blood miRNA Kit provide optimal binding conditions for several RNA species. The total RNA isolated includes mRNA and RNA molecules smaller than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, tRNAs, and miRNAs (see figure "High yields of small RNA species"). The chemistry of the PAXgene Blood miRNA Kit is complimentary to various downstream applications, including QIAGEN's PCR technologies. The obtained high quality miRNA can be analyzed using the QIAGEN miScript PCR System* (see figure "Analysis of miRNA species") or miRCURY LNA miRNA Probe PCR System

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 isolation 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 (see figure "PAXgene Blood miRNA procedure").

Applications

The isolated 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

Supporting data and figures

Analysis of miRNA species.

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 or the PAXgene Blood miRNA Kit, as indicated. miRNA species were analyzed using the QIAGEN miScript PCR System, with the indicated miScript Primer Assays. The lower CT values for RNA purified using the PAXgene Blood miRNA Kit indicate higher amounts of those miRNA species.

Specifications