Lipofection Sample Clauses

Lipofection. Using 12 well plates, 1.2 x 105 MSC per well were seeded with 500 μl of normal medium containing 10% foetal bovine serum (Biosera, South America Origin, Cat no: FB-1001G). Master mix consists of 3 μl of transfection reagent called Attractene (Qiagen, Hilden, Germany), 200 μl of serum free culture medium and optimised amount of inhibitor, mimic or negative control per well was made and incubated for 5-10 minutes at room temperature and then added to the cells drop-wisely and incubated at 37°C in the presence of 5% CO2 for 24 hours. Next, transfection media was replaced by differentiation media or cells were lysed using Trizol for RNA extraction and further investigation. The experiment was repeated several times with different seeding densities of cells and inhibitor/mimic concentration (from low to high) to reach the optimised protocol described here. XxXXX-302 Inhibitor To knock down xxXXX-302 family, miScript xxXXX Inhibitor (Qiagen, Hilden, Germany) was used which is a synthetic single stranded, 2-OMe modified RNA oligos. Negative control (Qiagen, Hilden, Germany) is chemically synthesized, single-stranded, modified RNA that has no homology to any known mammalian gene, transfected into the cells in parallel with inhibitor. MiR-302 family inhibitor or negative control were added to master mix with the optimized final concentration of 5 nM per well. XxXXX-302 Mimic To test the effect of overexpression of miR-302 family members on MSCs and validate the data from knock-down experiments, xxXXX-302a and xxXXX-302b mimic were transfected into the cells by the lipofection protocol. MiR-302b Mimic miRCURY LNA™ microRNA mimics (Exiqon, Vedbaek, Denmark) were selected to induce individual miR-302b over expression in MSCs. By introducing xxXXX mimic into cells, the proportion of RISC for this particular xxXXX rises and the activity of xxXXX increases. miRCURY LNA xxXXX Mimic designed base on three RNA strands (Figure 2-1). One guide strand and two segmented LNA-enhanced passenger strand which is not able to be loaded into the RISC and ensure that the resulted elevated xxXXX activity is from guide strand only (Manufacture handbook). MSCs were transfected by negative control (Exiqon, Vedbaek, Denmark) along with miR-302b mimic. The optimised final concentration of both miR-302b mimic and negative control was 0.5 nM per well. MiR-302a Mimic miScript xxXXX Mimics (Qiagen, Hilden, Germany) are chemically synthesized, double- stranded RNAs which mimic naturally occurring...