RNA interference (RNAi) screening is a powerful technique used to identify genes involved in specific biological processes. Tet - 213 cells, a well - characterized cell line, offer an excellent model for such screenings. As a Tet - 213 cell supplier, I am well - versed in the process of performing RNAi screening on these cells. In this blog, I will guide you through the step - by - step process of conducting an RNAi screening on Tet - 213 cells.
1. Preparation of Tet - 213 Cells
Before starting the RNAi screening, it is crucial to have healthy and actively growing Tet - 213 cells. The cells should be cultured in an appropriate growth medium, typically a high - glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin - streptomycin, and other essential nutrients.
Maintain the cells in a humidified incubator at 37°C with 5% CO₂. Regularly monitor the cell density and morphology. When the cells reach about 70 - 80% confluency, they are ready for the next steps. It is important to passage the cells according to standard procedures to ensure their viability and growth characteristics.
2. Design and Selection of siRNAs
The success of an RNAi screening largely depends on the quality of the small interfering RNAs (siRNAs) used. siRNAs are short double - stranded RNA molecules that can specifically target and degrade complementary mRNA sequences, leading to gene silencing.
There are several factors to consider when designing siRNAs. First, the target sequence should be unique to the gene of interest to avoid off - target effects. Many online tools are available for siRNA design, such as the siRNA Wizard from Invitrogen. These tools use algorithms to predict the most effective siRNA sequences based on factors like GC content, secondary structure, and target accessibility.
It is also advisable to select at least three different siRNAs for each target gene to increase the likelihood of successful gene silencing. Commercially available siRNA libraries can be a convenient option for large - scale RNAi screenings. These libraries often contain pre - designed siRNAs targeting a wide range of genes.
3. Transfection of siRNAs into Tet - 213 Cells
Once the siRNAs are selected, the next step is to transfect them into the Tet - 213 cells. Transfection is the process of introducing foreign nucleic acids, such as siRNAs, into cells. There are several transfection methods available, including chemical - based transfection, electroporation, and viral - mediated transfection.
Chemical - based transfection is the most commonly used method for RNAi screening in Tet - 213 cells. Lipid - based transfection reagents, such as Lipofectamine RNAiMAX, are widely used due to their high transfection efficiency and low cytotoxicity.
Here is a general protocol for chemical - based transfection:
- Prepare the siRNA - transfection reagent complex: Dilute the siRNA in Opti - MEM reduced - serum medium. In a separate tube, dilute the transfection reagent in Opti - MEM. Then, combine the two solutions and incubate at room temperature for 15 - 20 minutes to allow the formation of the siRNA - transfection reagent complex.
- Add the complex to the cells: Remove the growth medium from the Tet - 213 cells and replace it with fresh Opti - MEM. Then, add the siRNA - transfection reagent complex to the cells and gently mix.
- Incubate the cells: Return the cells to the incubator and allow them to incubate for 24 - 72 hours, depending on the experimental requirements.
4. Validation of Gene Silencing
After transfection, it is essential to validate the gene silencing efficiency. This can be done at both the mRNA and protein levels.
mRNA Level
Quantitative real - time polymerase chain reaction (qRT - PCR) is a commonly used method to measure the mRNA levels of the target genes. Extract total RNA from the transfected Tet - 213 cells using a commercial RNA extraction kit, such as the RNeasy Mini Kit from Qiagen. Then, reverse - transcribe the RNA into cDNA using a reverse transcription kit. Finally, perform qRT - PCR using specific primers for the target gene and a reference gene, such as GAPDH.
The relative mRNA levels can be calculated using the 2^(-ΔΔCt) method. A significant decrease in the mRNA levels of the target gene compared to the control cells indicates successful gene silencing.
Protein Level
Western blotting is a powerful technique for detecting and quantifying protein levels. Lyse the transfected cells using a suitable lysis buffer and separate the proteins by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS - PAGE). Transfer the proteins to a nitrocellulose or PVDF membrane and incubate the membrane with a primary antibody specific to the target protein. Then, use a secondary antibody conjugated to a horseradish peroxidase (HRP) or other detection tags. Visualize the protein bands using chemiluminescence or other detection methods.
A decrease in the intensity of the protein band corresponding to the target protein compared to the control cells confirms the gene silencing at the protein level.
5. Phenotypic Analysis
After validating gene silencing, the next step is to perform phenotypic analysis to determine the biological function of the target genes. The phenotypic analysis can vary depending on the research question.
For example, if you are interested in cell proliferation, you can use methods such as the MTT assay or the BrdU incorporation assay to measure the growth rate of the transfected cells. If you are studying cell migration, you can use the scratch assay or the transwell migration assay.
In addition to these traditional assays, high - content screening (HCS) technologies can be used for more comprehensive phenotypic analysis. HCS allows the simultaneous measurement of multiple cellular parameters, such as cell morphology, protein localization, and gene expression, using automated microscopy and image analysis software.
6. Data Analysis and Interpretation
Once the phenotypic data is collected, it needs to be analyzed and interpreted. First, normalize the data to account for variations in cell number, transfection efficiency, and other experimental factors.
Statistical analysis is then performed to determine the significance of the observed phenotypic changes. Commonly used statistical tests include the t - test, ANOVA, and chi - square test, depending on the type of data and experimental design.
Identify the genes whose silencing leads to significant phenotypic changes. These genes are likely to be involved in the biological process under study. Further validation and functional characterization of these genes can be carried out using additional experiments, such as rescue experiments or overexpression studies.
7. Considerations and Troubleshooting
During the RNAi screening process, several issues may arise. Here are some common considerations and troubleshooting tips:
Off - target Effects
Off - target effects occur when siRNAs target unintended genes, leading to false - positive results. To minimize off - target effects, use multiple siRNAs for each target gene and perform appropriate controls, such as non - targeting siRNAs.
Transfection Efficiency
Low transfection efficiency can result in poor gene silencing and inaccurate phenotypic analysis. Optimize the transfection conditions, such as the amount of siRNA, the ratio of siRNA to transfection reagent, and the transfection time. You can also use positive control siRNAs to monitor the transfection efficiency.
Cell Toxicity
Some transfection reagents and siRNAs may cause cell toxicity, which can affect the phenotypic analysis. Test different transfection reagents and optimize the transfection conditions to minimize cell toxicity. Monitor the cell viability during the transfection process using methods such as trypan blue exclusion assay.
Conclusion
Performing an RNAi screening on Tet - 213 cells is a complex but rewarding process. By following the steps outlined above, you can successfully identify genes involved in specific biological processes. As a Tet - 213 cell supplier, we are committed to providing high - quality cells and technical support to help you achieve your research goals.
If you are interested in purchasing Tet - 213 cells for your RNAi screening experiments or have any questions about the process, please feel free to contact us for further discussion and procurement. We look forward to working with you to advance your scientific research.
References
- Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21 - nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411(6836), 494 - 498.
- Hannon, G. J. (2002). RNA interference. Nature, 418(6894), 244 - 251.
- Zamore, P. D., Tuschl, T., Sharp, P. A., & Bartel, D. P. (2000). RNAi: double - stranded RNA directs the ATP - dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101(1), 25 - 33.
During the process of RNAi screening, it is also important to note that the use of appropriate peptides can sometimes assist in the study. For example, OVA Peptide (323 - 339), Prolactin - Releasing Peptide (1 - 31) (rat), and Enterostatin (bovine, Canine, Porcine) can be used in related immunological or physiological studies in combination with the RNAi screening on Tet - 213 cells. These peptides may help in understanding the downstream effects of gene silencing in a more comprehensive way.