Cell Culture Research Lab Report
The 14 sessions I have spent in the lab were focused on studying the mTOR pathway, its crucial role in normal cell functioning and cancer, the types of mTOR complexes found in cells, and their corresponding subunits. In particular, I have studied mlst8 silencing in a series of cancer cell lines as a potentially promising target for tumor treatment. As part of this project, I had the opportunity to learn about a range of laboratory techniques that constitute the fundamental pylons of cell and molecular biology research. Herein, I am providing a short description of a series of such methods and their use in research.
Cell culture represents the growth (often, for many generations) of animal or plant derived cells in an artificially maintained environment, with the addition of the necessary growth factors, signaling molecules, and nutrients as required. Importantly, cell lines originating from primary cultures (i.e. cultures obtained after isolating cells from the source organism) can usually proliferate a limited number of generations, so they must be used judiciously and occasionally renewed. The nutrients required for growing different cell types vary greatly, with more common ones being carbohydrates, aminoacids, vitamins, salts and ions, gases like oxygen and CO2, hormones, growth factors (the last two are very specific). All these are added to the liquid medium in which cells grow, usually in flat dishes that are kept in a controlled environment with strict values of pH, temperature, oxygenation. While in most cases cells can only be grown on a physical support, there are also suspension cultures, where the cells are floating in a liquid with all the nutrients. In terms of procedures that are often performed on cell cultures, one can mention, passages – transferring a fraction of the primary culture to a new vessel with a fresh liquid medium. Freezing in liquid nitrogen (i.e. cryopreservation), following a treatment with cryoprotectants like glycerol, is used for the long term storage of cells. The utility of cell cultures is gigantic, since they provide the raw material for most of the research carried on complex model organisms and allow all biochemical, genetic, and molecular biology studies and manipulations.
RNA extraction from cells is often the first step in a research pipeline aimed at assessing the expression level of genes or at studying the RNA variants in a cell. Ribonucleases in the cytoplasm, which come in contact with the RNA following cell lysis, makes this a rather delicate procedure, which requires cell lysis to be performed in liquid nitrogen or with RNAse inhibitors. Cell RNAses can prove remarkably difficult to neutralize and in addition to this, the external environment itself can be a source of RNAses. Another problem with RNA extraction is the mechanical fragility of RNA in contrast to DNA, hence, all manipulations must be careful to avoid shearing. The most common methods for RNA extraction are the guanidinium thiocyanate-phenol-chloroform method and column based purification, although the first one yields higher quantities and purity. First of all, a phase separation …