Gene Regulation

• Q1: What are the factors that affect siRNA transfection efficiency?

  A: The siRNA transfection efficiency is not ideal. Common reasons include the following aspects:

  1) The ratio of RNA to transfection reagent is not good. Due to differences in RNA sequences, different synthesis conditions, and whether it is labeled with fluorescence or other markers, RNA and transfection reagents will have different optimal conditions under different circumstances. It is recommended to conduct preliminary experiments first. optimization.

  2) The cell density is not good. Adjust the cell density to a confluency of 20-40% during transfection. Cells that are successfully transfected with siRNA will have down-regulation of target gene expression, but cells that are not successfully transfected will not be affected. At this time, the transfection efficiency and the total number of cells are very important. Generally, the transfection efficiency is high when the number of cells is small. Due to the timeliness of siRNA silencing, qRT-PCR detection can only be performed 48 hours after transfection, and protein detection can only be performed 48-72 hours after transfection. If the plating density is high during transfection, on the one hand, the transfection effect of the cells will not be ideal, which directly affects the silencing effect and data reliability. On the other hand, after 48 hours or even longer, when the optimal point of silencing detection is reached, the cells will be too dense. will affect the cell state and thus the experimental results.

  3) The RNA efficiency is not high. Select the optimal RNA during the experiment and use known efficient RNA as a control. When synthesizing siRNA, attention must be paid to selecting high-purity siRNA. The purity of siRNA is directly related to transfection efficiency and silencing efficiency.

  4) If the transfection reagent is not suitable, replace the transfection reagent, or choose a reagent specifically designed for siRNA transfection, such as Entranster reagent.

• Q2: What factors need to be considered when designing stable strain construction experiments?

  A: Several key factors that need to be considered in a stable integration test are:

   

  1) In most cases, low copy or even single copy can reduce the interference of artificial experimental factors in most cases;

  2) The probability of foreign gene integration not only determines the difficulty of screening stable strains, but also makes it easier to obtain mixed stable strains;

  3) The transcription activity of the integration site determines the expression quality of the foreign fragment in the stable strain;

  4) Stability after integration Different integration sites determine the stability of the exogenous fragment in the chromosome. Some areas are prone to recombination or loss, resulting in the loss of stable strains again;

  5) Use mixed stable strains or obtain multiple different monoclonal stable strains. Because stable integration is often accompanied by insertion and inactivation of host endogenous genes, you can use mixed stable strains or compare multiple monoclonal stable strains during experiments. Help obtain more accurate experimental data.

• Q3: How to improve RNA cell transfection efficiency?

  A：

  1) Purify RNA. Confirm the size and purity of RNA before transfection. To obtain high-purity RNA, it is recommended to use glass fiber to bind, elute or pass through 15-20% acrylamide gel to remove excess nucleotides, small oligonucleotides, proteins and salt ions in the reaction. Note: Chemically synthesized RNA usually requires gel electrophoresis purification (i.e. PAGE gel purification).

  2) Avoid RNase contamination. Trace amounts of RNase will cause RNA experiments to fail. Since RNase is ubiquitous in the experimental environment, such as skin, hair, all items touched by bare hands or exposed to the air, it is very important to ensure that every step of the experiment is free from RNase contamination.

  3) Healthy cell culture and strict operations ensure reproducibility of transfection. Generally, healthy cells have higher transfection efficiency. In addition, lower passage numbers ensure the stability of cells used in each experiment. In order to optimize the experiment, it is recommended to use transfected cells below 50 generations, otherwise the cell transfection efficiency will decrease significantly over time.

• Q4: What problems may arise during vector construction?

  A: Common experimental errors in vector construction:

  1) Using the wrong enzyme during double enzyme digestion;

  2) For double enzyme digestion, the two enzyme digestion sites are too close;

  3) The sticky ends generated by enzyme digestion can be complementary;

  4) Use methylation-sensitive restriction endonucleases, such as Dpnll;

  5) Improper selection of host bacteria, such as using a strain containing F plasmid in Gateway cloning;

  6) Use high copy number backbones to clone large fragments. For example, plasmids have a high relative copy number but the loading capacity is up to 30 kb, AAV has a minimum relative copy number of 4.7 kb and a high relative copy number, and lentivirus has a relative copy number but the loading can be stable at 9,2 kb.

• Q5: Which method is better, RNAi-mediated gene downregulation or CRISPR genome editing-mediated gene knockout?
  A: It depends on the specific experimental purpose. Among them, RNAi-mediated gene downregulation is more suitable than genome editing when there is no need to change the genetic code. However, to create a truly null allele on a chromosome, CRISPR genome editing is better.