Learning Center

• Q1: What are the factors that affect RNA interference efficiency?

  A：

  ①Differences in structure (double-stranded RNA has higher potency than single-stranded RNA), Similar behavior of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway ( https://academic.oup.com/nar/article /31/9/2401/1080348)(http://europepmc.org/article/PMC/154224 ) ; _

  ②Difference in sequence,

  ③Differences in mRNA position, https://www.biomart.cn/experiment/430/443/450/680/2282559.htm

• 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) The copy number of the exogenous inserted fragment. In most cases, low copy or even single copy can reduce the interference of human experimental factors;

  2) The probability of integration, which 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 exogenous fragments in the chromosome. Some regions 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 insertional inactivation of host endogenous genes, using mixed stable strains or comparing multiple monoclonal stable strains during experiments can help obtain more accurate experimental data. ( https://wenku.baidu.com/view/306486dc6f1aff00bed51e78.html )

• Q3: What are the factors that affect siRNA transfection efficiency?

  A: ( https://zhuanlan.zhihu.com/p/100352316 )

• Q4: How to solve the problem of high toxicity of lipofectamine transfection reagent?

  A: https://zhuanlan.zhihu.com/p/29140323

• Q5: In virus-infected cell experiments, what is the reason for cytotoxicity when using infection enhancers?

  A：

  ① If the dosage of enhancer is too large, its dosage can be appropriately reduced;

  ② If the amount of virus is too large, the amount of virus can be appropriately reduced, and the method of changing the culture medium or increasing the amount of culture medium can be used. ( https://zhuanlan.zhihu.com/p/126995361 )

• Q6: What is the appropriate amount of cell inoculation for virus infection?
  A: Determine the inoculation amount based on the cell proliferation rate. Generally, it is necessary to ensure that the cells almost cover the bottom of the culture dish about 4 days after infection. For most cell lines: the passage period is 2-3 days, and the cell plating density during infection should be maintained within the range of 20%-30%. For some primary cells: cells grow slowly, and the confluency can be increased to 50%-60% during inoculation, ensuring that the cell confluence reaches 90%-100% about 4 days after infection. For non-dividing cells that no longer proliferate after inoculation (such as neuronal cells), inoculate at 100% confluence.
• Q7: After adding the virus, the cells died seriously. What is the reason? How to deal with it?
  A: Generally, viruses have certain toxicity to cells, and the infection MOI can be adjusted and lowered. And pay close attention to the cell status, and observe the cell status regularly at 4, 8, and 12 hours after infection. If the cell status deteriorates, immediately change the medium of the cells and replace the virus-infected culture medium with fresh complete culture medium.
• Q8: How to improve virus infection efficiency?
  A: The efficiency of virus infection is affected by many factors, including the growth status of the cells themselves, the number of cells, and the ease of infection of the cells. Therefore, during the experiment, ensuring normal cell proliferation and clear outlines while maintaining appropriate cell density and selecting optimal infection conditions can better ensure infection efficiency. For suspension cells, centrifugal infection can be used to reduce the volume of virus infection and thereby improve the infection efficiency. If the culture plate is sealed, centrifuge it with a square-angle rotor centrifuge at 1000 g for 1 hr, and then put it back into the incubator to continue normal culture.
• Q9: Cells can be infected by viruses, but why is GFP fluorescence so weak?
  A: After the GFP virus infects cells, the fluorescence intensity of the cells depends on the number of virus particles entering the cells, the proliferation status of the cells themselves, cell type, observation time, GFP gene promoter activity and other factors. Therefore, the more viral particles the target cells are infected with and the faster the cells proliferate, the stronger the GFP fluorescence will be. Generally, GFP protein expression reaches its peak 96-120 hrs after virus infection in cells that proliferate quickly; in cells that proliferate slowly, this time is longer. The fluorescent expression of the GFP gene behind a strong promoter is strong, and conversely, the fluorescent expression behind a weak promoter is weak.
• Q10: What are the factors affecting cell transfection experiments?

  A: https://zhuanlan.zhihu.com/p/29100441

• Q11: How is the time specificity of conditional gene knockout achieved?

  A：https://www.sohu.com/a/200476821_100007412