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
Purification begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and 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 automated RNA purification protocol consists of 2 parts (or protocols), "PAXgene Blood RNA Part A" and "PAXgene Blood RNA Part B", with a brief manual intervention between the 2 parts.
The centrifuged, washed, and resuspended nucleic acid pellet is 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.
Average sample preparation time (based on data from 12 sample preps) is 125 minutes, with only approximately 20 minutes of hands-on time.