For purification of miRNA and total RNA from whole blood
Integrated system for collection, stabilization, and purification
Effective purification of miRNA and total RNA
RNA stabilization for up to 3 days at 18–25°C
Stabilization for at least 50 months at –20°C or –70°C
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. Used with the tubes, the kit provides a complete preanalytical solution for collection and stabilization through to purification of high-quality total RNA, including miRNA, for molecular analysis.
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
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.
High yields of small RNA 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 (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.
The QIAcube.
Samples can be processed manually or automated on the QIAcube.
Performance
Blood samples collected in PAXgene Blood RNA Tubes can be safely stored or transported at 15–25°C for up to 72 hours, at 2–8°C for up to 5 days, or at –20°C or –70°C for at least 50 months without showing any significant RNA degradation or changes in transcript levels. Total RNA >18 nucleotides is purified from the stabilized blood samples with the PAXgene Blood miRNA Kit and includes both mRNA and small RNAs such as miRNA (see figure "High yields of small RNA species"). The miRNA species can be analyzed using the QIAGEN miScript PCR System (see figure "Analysis of miRNA species") or other real-time RT-PCR systems.
Principle
Blood samples are collected in PAXgene Blood RNA Tubes. The tubes contain a reagent that lyses blood cells and immediately stabilizes intracellular RNA to preserve the gene expression profile. RNA stabilization is critical for reliable downstream gene expression analysis. Without stabilization, degradation of RNA and upregulation or downregulation of transcripts occur immediately after blood is drawn.
Procedure
With the PAXgene Blood miRNA Kit, total RNA >18 nucleotides (including miRNA) is purified from the stabilized blood samples using well-established PAXgene silica-membrane technology. Purification can be carried out in a manual format or it is automatable on the QIAcube (see figure "The QIAcube").
Applications
The purified total RNA is ready to use and is ideally suited for any downstream application, including:
RT-PCR and real-time RT-PCR
Differential display
cDNA synthesis
Northern, dot, and slot blot analyses
Primer extension
Poly A+ RNA selection
RNase/S1 nuclease protection
Microarray analysis
Features
Specifications
Applications
RT-PCR and real-time RT-PCR, differential display, cDNA synthesis, northern, dot, and slot blot analyses, primer extension, microarray analysis
2.5 ml (100 mg/ml; 7000 units/ml, solution)
Unit definition: That amount of enzyme causing the hydrolysis of RNA at a rate such that k (velocity constant) equals unity (Kunitz units) at 25°C and pH 5.0.
