A comparative research on planktons separated from liver by different density gradient centrifugation with Percoll in rabbits

Objective To experimentally evaluate the effect of different density gradient centrifugation on plankton separation from tissues with Percoll.Methods Mixture of three algae species with the homogenate of rabbit liver were divided into 5 centrifugation groups: well-mixed homogenate at high-speed centrifugation,well-mixed homogenate at low-speed centrifugation,overlaying homogenate at high-speed centrifugation,overla ying homogenate at low-speed centrifugation,and mixted homogenate centrifugated at 12 000r/min as control group.The planktons were separated from the mixture by density gradient centrifugation with Percoll.After separation,the sediment of different layer was detected under microscope,and DNA was extracted.The specific fragment of algae 16S rDNA and chlorophyll-related gene were subsequently amplified by PCR.Results In well-mixed homogenate at high-speed centrifugation,the algae were localized in the middle of tube as stratified layers.In overlaying homogenate at high-speed centrifugation,algae were detected beneath the tissue layer.In homogenate at low-speed centrifugation,less algae were found closely under the layer of tissue,and for the control,algae was in the bottom of tube.With DNA analysis,a 447bp fragment of 16S rDNA and a 194bp fragment of chlorophyll-related gene were detected in sediments separated from all group with Percoll.However,in control homogenate mixed with algae,no amplified product was detected with darker background of electrophoresis gel because of the interference of tissue DNA.Conclusion It is recommended that tissues homogenate should be centrifugated at high-speed density gradient centrifugation with Percoll,and sediments beneath the tissue layer should be collected for plankton isolation.