DNA/RNA EXTRACTION
Once the environmental samples of interest have been collected, it is time for the extraction of nucleic acids. This is a process where the DNA and RNA molecules are separated from the rest of cellular components, such as proteins and fatty acids. The result is a pure solution of either DNA or RNA molecules.
Different commercial extraction kits are commonly used, but the nucleic acid extraction basically consists of:
Cell lysis and sample homogenization
The first step is to break down the cells that contain the DNA and RNA molecules. This can be a problematic process in the case of bacterial cells as they can be hard to lyse, particularly gram-positive bacteria, due to a rather resistant cell wall. For this reason, the so-called lysis buffers are typically use. Modern versions usually contain guanidinium thiocyanate, which disrupts the cells, deactivates nucleases and improves the stabilization of DNA and RNA. But even lysis buffers are not sufficient and, therefore, homogenization by ultrafast mechanical vibrations is highly advisable to ensure the lysis of a significant fraction of the cells. OmiBox makes use of the DNA/RNA lysis buffer from Zymo Research, and the MonoLyser homogenizer for a quick and efficient cell lysis.
DNA/RNA isolation
There are several commercial kits for the extraction of DNA and RNA from environmental samples. In OmiBox, we usually employ Zymo DNA/RNA co-isolation kits, with which we can reduce the elution volume down to 6 µl to maximize the concentration of the final isolate. This is particularly important for water samples collected onto membrane filters, which sometimes may contain a very limited amount of biomass. These commercial extraction kits are based on the spin column technique, for which OmiBox makes use of the portable centrifuge integrated into BentoLab compact laboratory. The entire workflow is done inside an isolation chamber where filtered air is pumped in to ensure a sterile environment.
DNA/RNA measurements
The final step before the library preparation is to measure the concentration of DNA and RNA and confirm whether we have been successful with the extraction procedure. OmiBox integrates the Qubit 4, a highly portable fluorometer for quick and precise measurements of the concentration (and even integrity) of DNA and RNA molecules. Only 1 µl is enough, which is particularly valuable to save the usually scarce RNA for the library preparation.
