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Overview

Hysigen provides an extensive array of shRNA reagents to furnish you with optimal tools for your RNAi experiments. We specialize in packaging all major virus types, including lentivirus, AAV, and adenovirus, across various titer scales, facilitating the delivery of your shRNAs into difficult-to-transfect cells.

Key Advancements

• Abundant collections of vector backbones and components.

• 100% sequence validation, ensuring accuracy, with rapid turnaround times and competitive pricing.

• Robust technical support for shRNA selection, vector design, and troubleshooting, enhancing the efficiency of your experiments.

Workflow

1. Cell Preparation

Cell resuscitation culture, using cells with a fast proliferation rate, cells capable of forming good monoclonal clones, and cells with high transfection efficiency are the key factors for the success of the experiment, and it is recommended to meet the following conditions:

- Resuscitated cells should be passaged more than 3 times;

- Use cells with good clone formation as much as possible;

- Passaging of cells 2 days before transfection;

- Change the cell culture medium one day before transfection;

- Keep the cells at 50-85% confluence before transfection to maintain   the cells in the best proliferation state.

2. shRNA + SSR transfection (or Transduction, Depends on the vector type )

- Cell density is 70-85%.

- Mix shRNA plasmid+lipo and add to cells.

- After incubation at (37 ºC, 5% CO₂) overnight.

- Observe the GFP expression rate in cell after 48 hours; the positive rate of the GFP control should be more than 50%.

Note: All the media used in the transfection process are antibiotic-free media; otherwise, it will lead to cell death.

3. Transfection and Drug Screening

After 48h of transfection, the cells are screened with Puromycin (Puro); Once cell confluence reaches 80%, passage the cells at a 1:2–1:3 ratio and continue drug screening for 1–2 weeks. This completes the construction of the stable transfected cell line.

Technical Information

RNA Interference (RNAi) is the process of effectively silencing or inhibiting the expression of a target gene, which is achieved by degrading the corresponding mRNA of the target gene using double-stranded RNA (dsRNA).