COMPARISON OF GILL MICROBIAL DNA ISOLATION METHODS IN Oncorhynchus mykiss FOR APPLICATION IN MICROBIOME RESEARCH
Microbiome research- the study of the microorganisms that live on and within a host- has been applied to aquaculture to better understand the role that bacteria play in host health. While much research has been conducted on the gut microbiota of several fish species, less priority has been given to studying microbial communities associated with other mucosal surfaces such as the skin and gills. Furthermore, due to the low abundances of microorganisms in some of these mucosal surfaces, isolating a sufficient amount of quality DNA for microbiome analyses has proven to be a challenge for the field. Failing to thoroughly isolate a representative sample of bacterial DNA can result in mischaracterizing or underestimating microbial diversity. In this study, five different isolation procedures were conducted to compare microbial DNA yield from rainbow trout gill tissue. In the first group, four gill arches were vortexed in 30mL PBS and removed. Subsequent centrifugation steps at high and low speed removed trout cells and pelleted bacterial cells respectively. Pelleted cells were washed in PBS and added the Qiagen PowerSoil Isolation Kit (PS kit). In the second and third group, four gill arches were vortexed and then removed in either 30mL of PBS or NaCl respectively. The vortexed solutions were then spun at a low speed to remove trout DNA. The supernatant was put through a 0.45µm and then a 0.22µm filter subsequently. Finally, the 0.22µm filter was added to the Qiagen PowerWater Isolation kit (PW kit). In the fourth group, a swab of the gill mucus from four gill arches was added directly to the PS kit. In the fifth group, 1cm2 gill tissue was added directly to the PS kit. DNA yields will be measured using a NanoDrop spectrophotometer and qPCR of the 16s rRNA gene will determine the success of bacterial DNA isolation. In addition, qPCR will be conducted for rainbow trout DNA to indicate relative amounts of bacterial to host DNA abundance.
