The PAXgene 96 Blood RNA Kit is for high-throughput purification of cellular RNA in a manual format. Used with PAXgene Blood RNA Tubes (available from BD, cat. no. 762165) for sample collection and stabilization, the system provides a complete preanalytical solution for collection and stabilization through to purification of high-quality RNA to support high-throughput projects, such as clinical research studies.
The PAXgene 96 Blood RNA Kit allows high-throughput isolation of total RNA from human whole blood. Together with PAXgene Blood RNA Tubes (available from BD, cat. no. 762165), it provides a complete preanalytical solution for collection and stabilization through to purification of high-quality RNA. The robust PAXgene 96 Blood RNA procedure is highly reproducible and typically yields between 4 and 20 µg of RNA from 2.5 ml human whole blood with minimal risk of sample-to-sample cross contamination. PAXgene Blood RNA Tubes contain a proprietary blend of reagents that bring about immediate stabilization of RNA. This blend prevents the drastic changes in the cellular RNA expression profiles that normally take place in vitro, after blood collection (see figures " RNA isolation from blood using standard methods", " Stabilization of gene expression", and " Detection of tyrosinase following blood storage"). The high-throughput procedure is simple, fast, and efficient, and processing 96 samples in parallel provides significant time savings in your clinical research studies.
In the PAXgene 96 Blood RNA System, blood is collected directly into PAXgene Blood RNA Tubes, and RNA is isolated using the PAXgene 96 Blood RNA Kit. PAXgene Blood RNA Tubes contain a proprietary blend of reagents that bring about immediate stabilization of RNA. Blood samples can be stored in the PAXgene Blood RNA Tubes for up to 5 days at room temperature. RNA is then isolated using silica membrane technology supplied in the PAXgene 96 Blood RNA Kit. The resulting RNA accurately represents the expression profile in vivo and is suitable for use in a range of downstream applications.
Blood samples (2.5 ml) are collected in PAXgene Blood RNA Tubes (available from BD, cat. no. 762165), and may be stored or transported at room temperature if desired. In the PAXgene 96 Blood RNA procedure, RNA isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, and proteinase K is added to digest proteins. Alcohol is added to adjust binding conditions. Lysates are applied to the PAXgene 96 Filter Plate and centrifuged to remove cell debris. The lysate is then applied to a PAXgene 96 RNA Plate (see flowchart " PAXgene 96 Blood RNA procedure"). Residual DNA is removed through a DNase I digestion. Remaining contaminants are removed in three efficient wash steps before pure RNA is eluted.
The RNA isolated from human whole blood is ready for use in a wide range of downstream applications, including:
Blood was collected and RNA was isolated using standard methods (collection in EDTA tubes; no stabilization; RNA isolation using a guanidinium-based method). The graph shows changes in expression of 12 genes after blood collection, measured using real-time RT-PCR. (Data kindly provided by Source Precision Medicine, Boulder, Colorado, USA.)
|Applications||PCR, real-time PCR, qPCR, NASBA, cDNA synthesis|
|Time per run or per prep||<3.5 hours|
|Elution volume||90–120 µl|
|Main sample type||Whole blood|
|Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein||Cellular RNA|
|Sample amount||2.5 ml|
|CE/FDA/IVD compatible||Research use only|
|Processing||Manual (centrifugation or vacuum)|
Several kit options are available for this application. We recommend using the PAXgene Blood RNA System, which enables the collection, stabilization and transportation of 2.5 ml human whole blood samples, and subsequent rapid and efficient isolation of cellular RNA.
The PAXgene 96 Blood RNA Kit uses a combination of vacuum and centrifugation to isolate RNA. The vacuum manifold required is the QIAGEN QIAvac 96 vacuum manifold, catalog # 19504. The suggested centrifuge is manufactured by Sigma and sold by QIAGEN, catalog # 81010, as is the additional plate rotor for spinning 2 x 96 plates, catalog # 81031. Additionally, the final heating step of the protocol utilizes an alloy block, the PAXgene 96 Incubation block, catalog # 9238279. Please contact QIAGEN technical service (www.qiagen.com) for more information.