Description
The PAXgene Blood RNA System consists of a blood collection tube (PAXgene Blood RNA Tube) and a nucleic acid purification kit (PAXgene Blood RNA Kit). It is intended for the collection, storage and transport of blood and stabilization of intracellular RNA in a closed tube, and subsequent isolation and purification of host RNA from whole blood for RT-PCR used in molecular diagnostic testing. Performance characteristics for the PAXgene Blood RNA System have only been established with FOS* and IL1B** gene transcripts. The user is responsible for establishing appropriate PAXgene Blood RNA System performance characteristics for other target transcripts. *FOS is the gene symbol for human Fos Proto-Oncogene, AP-1 Transcription Factor Subunit. **IL1B is the gene symbol for human Interleukin 1 Beta.
Total RNA purified using the PAXgene Blood RNA System is highly pure, with A 260 / A 280 values between 1.8 and 2.2 and ≤1.0% (w/w) genomic DNA in ≥95% of all samples processed (see "RNA yield, purity and genomic DNA contamination - automated processing."). At least 95% of samples show no inhibition in RT-PCR, when eluate accounts for up to 30% of the RT-PCR reaction. RNA yields from 2.5 ml healthy human whole blood are ≥3 µg for ≥95% of the samples processed. Since yields are highly donor-dependent, individual yields may vary. For individual donors, the PAXgene Blood RNA System provides highly reproducible and repeatable yields (see figures "Reproducible and repeatable RNA purification "and "Repeatability and reproducibility of RNA yield"), making the system highly robust for clinical diagnostic tests. The automated protocol of RNA purification using the PAXgene Blood RNA System provides pure RNA in high yield with reproducible and repeatable RT-PCR results, as shown in the figures (see "RNA yield, purity and genomic DNA contamination - automated processing.").
Copy numbers of individual RNA species in blood can change significantly during sample 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 silica-membrane technology in spin column format, and when combined with PAXgene Blood RNA Tubes, the PAXgene Blood RNA Kit provides a rapid procedure and the chemistry to isolate RNA of high quality and purity.
The PAXgene Blood RNA procedure is simple and can be performed using manual or automated procedures (see flowcharts "Manual PAXgene Blood RNA procedure "and "Automated PAXgene Blood RNA procedure"). Manual PAXgene Blood RNA procedure The PAXgene Blood RNA procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, resuspended, and incubated in optimized buffers together with proteinase K to bring about protein digestion. An additional centrifugation through the PAXgene Shredder spin column is carried out to homogenize the cell lysate and remove residual cell debris, and the supernatant of the flow-through fraction is transferred to a fresh microcentrifuge tube. Ethanol is added to adjust binding conditions, and the lysate is applied to a PAXgene RNA spin column. During a brief centrifugation, RNA is selectively bound to the PAXgene silica membrane as contaminants pass through. Remaining contaminants are removed in several efficient wash steps. Between the first and second wash steps, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA is eluted in elution buffer and heat-denatured. Automated PAXgene Blood RNA procedure Sample preparation, automated on the QIAcube Connect MDx, follows the same steps as the manual procedure, enabling you to continue using the PAXgene Blood RNA Kit for purification of high quality RNA. The procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, resuspended, and transferred from the PAXgene Blood RNA Tube into processing tubes, which are placed into the thermoshaker unit on the QIAcube Connect MDx worktable. The operator selects and starts the "PAXgene Blood RNA Part A "protocol from the menu. The QIAcube Connect MDx performs the steps of the protocol through to elution of RNA in elution buffer. The operator transfers the microcentrifuge tubes, containing the purified RNA, into the thermoshaker unit of the QIAcube Connect MDx. The operator selects and starts the "PAXgene Blood RNA Part B "protocol from the menu, and heat denaturation is performed by the QIAcube Connect MDx. Average sample preparation time (based on data for 12 sample preps) is 151 minutes, with only approximately 20 minutes of hands-on time.
