Optimal Transfection Conditions
Researchers have a plethora of options when it comes to selecting an optimal transfection reagent to facilitate cellular uptake of nucleic acids (such as proteins, siRNA, DNA and miRNA). In addition to general purpose transfection reagents, many cell line specific reagents are also commercially available. As the name indicates, cell line specific transfection reagents are pre-optimized for cell lines, which are established immortal cultures of a specific cell type.
The basic objective of transfection experiments is to study cellular processes that trigger protein synthesis. This can be accomplished in a transient or stable transfection. The gathered data can be used for various purposes, from gene therapy to plant and livestock production. To reach end goals quickly and efficiently, scientists need to set up optimal transfection reactions every time.
Cell lines thrive well and propagate when grown in favorable living conditions. Examples of cell lines include hamster ovarian cells (CHO), human pancreatic cells (AsPC-1), human neuroblastoma cells (CLBPEC), human umbilical vein endothelial cells (HUVEC), cord blood stem cells, human ovarian cancer cells (SKOV3), human hepatoma cells (HUH7) and many others.
Optimal Transfection Reagents
Using the appropriate transfection reagent increases the efficiency of transfection and limits the amount of cytotoxicity. There are several pre-optimized transfection reagents available on the commercial market. Cell line specific transfection reagents come with protocols to assist researchers in establishing efficient transfections with minimal toxicity.
Data reveals that the cell membrane phospholipid bilayer plays an important role in restricting exogenous DNA or RNA molecules from entering the cell. Successful transfection depends on several factors, such as preferred reagents, the protocol used, type of cells to be transfected and desired outcome of the transfection process. Some cells are inherently difficult to transfect, such as primary cells or suspension cells. Some cell types can exhibit better transfection efficiency when transfected in a certain way, such as reverse transfection versus forward transfection. Off-target effects and cytotoxicity of the transfection system often leads to low transfection efficiency.
Cell lines may undergo significant changes as the culture matures, confluency increases and nutritional medium becomes scarce. As a result, the efficiency at which they can be transfected varies. Scientists have observed that cells may show poor transfection results if they undergo the process soon after being thawed from cryopreservation. The same cells may exhibit better transfection efficiency after recovering completely from the effects of cryopreservation. Good laboratory practice is to use cell lines purchased from authorized sources, such as DSMZ and ATCC, and to optimize the cell line growth prior to starting any transfection process. In addition, a number of transfection services are commercially available from several companies.
Many protocols are available and already optimized to achieve the most efficient, viable anD reproducible performance across multiple cell types. If these protocols do not work, cell-specific protocols may be a better choice to optimize your experiment. CROs such as Altogen Labs employ experienced scientists who can recommend optimal transfection reagents and procedures for different cell lines.
Different transfection reagents, whether general purpose or cell line specific, work differently to insert foreign DNA, siRNA or protein into cultured cells. While some reagents work by shrinking the nucleic acid to be transfected, others are particularly effective in the serum contained in cell cultures. Certain transfection reagents have been designed with positively charged molecular linkers such that external nucleic acids readily coil around these molecular arms and then enter cells when the reagent molecules are endocytosed.