The NucleoSpin RNA Stool kit is designed for efficient isolation of RNA, including small RNA species, from fresh and frozen stool samples. The kit combines NucleoZol with Lysis Buffer RST1 for chemical disruption of stool and associated microbes. The mechanical lysis is performed using NucleoSpin Bead Tubes Type A (containing ceramic beads) in combination with a mechanical disruption device. No enzymatic reactions, such as protease digestion, are required to homogenize the sample material.
Undissolved sample material and the ceramic beads are subsequently removed by a short centrifugation. A subsequent lysate clearing step comprises addition of Binding Buffer RST2 followed by a short centrifugation. The supernatant is finally cleared by passing it through a NucleoSpin Inhibitor Removal Column, which removes substances in stool samples that interfere with qPCR. Binding conditions are adjusted by adding additional Binding Buffer RST2 to the flowthrough of the NucleoSpin Inhibitor Removal Column. Next, the sample is loaded onto a NucleoSpin RNA Stool Column. Residual contaminants such as complex polysaccharides, bile salts, and other PCR inhibitors are removed by an efficient washing procedure using Buffer RST2, as well as Wash Buffers RST3, RST4, and RST5. In between the different washing steps, residual DNA is removed via an on-column DNA digestion step using rDNase. After a drying step, ready-to-use RNA can be eluted with RNase-free water.