Medição de níveis de mRNA ao longo do tempo durante a levedura s. cerevisiae hipóxica resposta

The overall goal of this experiment is to monitor the mRNA levels of all genes over time as S.Cerevisiae cells are exposed to hypoxia.This method can help answer key questions in the cell signaling fields such as what it is the kinetics of gene expression changes during a cellular response?The main advantage of this technique is that mRNA levels are measured over time as opposed to being measured at one point during the cellular response.Though this method can provide insight into the yeast hypoxic response, it can also be applied to other microbial species that can be grown in liquid culture.Visual demonstration of this method is critical for conveying the detailed layout of flasks and tubing connections and the rapid collection of cells from hypoxia.One day or more before the hypoxia time course prepare the materials needed.Place the nitrogen tank incubator, vacuum, and filtering system in close proximity to enable quick processing of cells.Prepare sterile liquid YPD media by mixing glucose yeast extract, and peptone in a glass bottle and autoclaving it.The next step is to plan the layout of the flasks in the incubator following this schematic, from the nitrogen tank the first flask will be a water trap.The second flask will be the last time point, the third flask will be the second to last time point and so on until the last flask, which is a final water trap.For example, here is a possible layout of flasks and connections for seven hypoxic time points.On the day before the time course prepare a pre-culture of one yeast strain.Use a sterile applicator stick to pick up a full colony from a plate, place the stick into 5 mL of liquid YPD in a sterile test tube and shake the stick until most of the cells are in suspension.Rotate the tube at 30