PAXgene Blood miRNA Kit
For purification of intracellular RNA, including miRNA, from whole blood
- Effective purification of intracellular RNA, including miRNA
- Standardized sample processing prior to analysis
- Integrated DNase treatment for removal of gDNA
- Purification can be automated on the QIAcube
|Sample amount||2.5 ml|
|Elution volume||80 µl|
|Main sample type||Whole blood|
|Time per run||
Manual: 90 min/12 samples
Automated: 125 min/12 samples
|Typical RNA yield||>3 µg/2.5 ml sample (95% samples processed)|
|RNA purity||1.8–2.2 (A260/A280)|
The PAXgene Blood miRNA Kit is intended for the purification of total RNA (including miRNA) from whole blood collected in PAXgene Blood RNA Tubes.
For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.
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.
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.
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 (see High yields of small RNA species). The miRNA species can be analyzed using the QIAGEN miScript PCR System (see Analysis of miRNA species) or other real-time RT-PCR systems.
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.
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 PAXgene Blood miRNA procedure).
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
PAXgene Blood miRNA Kit
1. Can the PAXgene Blood miRNA Kit be used for diagnostic or prognostic procedures?
No. The product is ‘For Research Use.’ Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
2. How great is the enrichment of miRNA when using the dedicated PAXgene Blood miRNA Kit compared to the PAXgene Blood RNA Kit?
If the PAXgene Blood miRNA Kit is used, an enrichment of small RNAs as part of the total RNA is achieved. The enrichment may vary between different miRNA species and samples, but on average an enrichment of about three or more C T -values can be expected.
3. How many samples can be processed with the PAXgene Blood miRNA Kit?
The kit can be used for 50 RNA isolations.
4. Which sample types can be processed using the PAXgene Blood miRNA protocols?
2.5 ml whole human blood collected in PAXgene Blood RNA Tubes.
5. Do the samples require pretreatment before starting the manual protocol or before loading onto the QIAcube instrument?
Yes. Samples have to be incubated at least 2 h at room temperature (18-22°C). Incubation of the PAXgene Blood RNA Tube overnight may increase yields slightly in some cases. Room temperature storage for up to three days has no effect on the RNA profile. During the preparation of the RNA the manual pretreatment consist of a centrifugation and a pellet resuspenion step (see the PAXgene Blood miRNA Handbook).
6. Do you have stability data for RNA isolated with PAXgene miRNA Kit?
Stability data are not yet available, but a stability study was started.
7. What are the expected yields of RNA from PAXgene Blood RNA Tubes isolated with the PAXgene Blood miRNA kit?
Typical yields of RNA isolated from 2.5 ml healthy, human, whole blood (4.8 x 106 –1.1 x 107 leukocytes/ml) are ≥3 μg for >95% of the samples processed (see Technical Note). Since yields are highly donor-dependent, individual yields may vary.
8. How should the samples be stored before purification of RNA?
Room temperature storage of the filled PAXgene Blood RNA Tubes for up to three days has no effect on the RNA profile. For longer storage they should be stored at –20°C or –70°C.
9. What is the maximum number of samples for the QIAcube variant of the PAXgene Blood miRNA protocol for a single run?
Up to 12 samples can be processed in a single run.
10. Why is the final heat denaturation step so important?
Denaturation of the eluate is essential for maximum efficiency in downstream applications, such as RT-PCR, other amplification reactions, or cDNA synthesis. It is not necessary to denature samples more than once; samples remain denatured after freezing and thawing.
11.Is it possible to isolate genomic DNA or virus nucleic acids with PAXgene Blood miRNA protocol?
No. The PAXgene Blood RNA System has been optimized for cellular RNA only. PreAnalytiX offers a dedicated system for collection and isolation of genomic DNA from human whole blood (see PAXgene Blood DNA System).
|Product||Catalog Nr.||Price (USD)|
PAXgene Blood miRNA Kit (50)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.