We recommend to start with 50–200 mg of stool. Homogenize each sample in a 2 ml bead beating tube containing a mixture of lysis beads. The mechanical collisions between beads combined with the chemical disruption of cell membranes allow for lysis of host and microbial cells. IRT is then used to remove substances that are commonly found in stool samples, which inhibit PCR and other downstream applications. Then pass the lysate through a MB RNA Spin Column. Remove the DNA using on-column DNase digestion. Then use a wash solution to remove the enzyme and any digested nucleic acids (see figure RNeasy PowerFecal Pro Procedure). Elute the RNA with RNase-free water. It is now ready to use in any downstream applications.
Sample storage and preservation
The yield and integrity of nucleic acids isolated from microbes in stool is greatly influenced by the state of the digestive system, diet of the individual, and the length of time between collection of the sample and preservation. To optimize the quality of nucleic acids from stool, process the sample as quickly as possible after collection. The PowerProtect DNA/RNA reagent stabilizes stool samples at room temperature.
Automated processing on the QIAcube Connect
RNA purification with the RNeasy PowerFecal Pro kit can be fully automated on QIAcube Connect or the classic QIAcube.
The QIAcube Connect instrument is designed for low-throughput automation of the sample preparation workflow based on spin column technology. The automated protocol follows the same steps as a manual procedure (i.e., lyse, bind, wash, and elute) processing up to 12 samples per run.
QIAcube Connect is designed to perform automated purification of nucleic acids and proteins in molecular biology applications. The system is intended for use by professional users trained in molecular biological techniques and the operation of QIAcube Connect.