How much siRNA do you use for transfection?

In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).

What is Lipofectamine RNAiMAX?

Lipofectamine® RNAiMAX is a proprietary RNAi-specific cationic lipid formulation designed specifically for the delivery of siRNA and miRNA into all cell types.

How do you dissolve siRNA?

General considerations: Dissolve siRNA in sterile water and prepare 5 µl aliquots, store at –80°C. For use, dilute siRNA in siRNA dilution buffer (diluted siRNA is not stable, use only once). 5x siRNA buffer aliquots (Dharmacon) are stored at 4°C in the cell culture lab.

How do you increase siRNA transfection efficiency?

9 Tips for Optimal siRNA Transfection

  1. Use the most appropriate siRNA concentration.
  2. Prepare a suitable siRNA stock solution.
  3. Transfect healthy cells.
  4. Check serum quality.
  5. Know the target gene in and out.
  6. Always use positive and negative controls.
  7. Follow up the transfection reagent protocol.

How does transfection reagent work?

Upon cellular uptake, transfection complexes are sequestrated into intracellular vesicles. Our transfection reagents are able to induce the release of the nucleic acids into the cytoplasm through vesicle membrane rupture or fusion.

How long does it take for siRNA to work?

Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA.

What is the difference between lipofectamine 2000 and rnaiMaX?

Lipofectamine® rnaiMaX reagent is designed specifically for the delivery of sirna and mirna while Lipofectamine® 2000 reagent delivers Dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays.

Can I dilute siRNA in water?

All Answers (4) SiRNA buffer is recommended for long term storage, you can resuspend your SiRNA in RNAse free molecular grade water (or 0.22µm filtered DEPC treated water) for immediate use.

How long does siRNA last?

The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA. Transfections may be repeated to maintain silencing.

How can I improve my siRNA knockdown?

Top 4 ways to make your siRNA experiment a success

  1. Choose appropriate siRNA reagents and delivery methods that are adapted to your cell type.
  2. Use appropriate positive and negative controls in each and every experiment.
  3. Optimize siRNA delivery conditions.
  4. Confirm your knockdown on the mRNA level using qPCR.

How long to incubate siRNA plasmid for transfection?

Mix gently by rocking the plate back and forth. Incubate the cells at 37°C in a CO 2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.

What happens when siRNA is inserted into a cell?

Soon after being inserted in the mammalian cell, the siRNA molecules become a part of the RNA-induced silencing complex (RISC). Guided by the antisense strand of the siRNA, RISC degrades the targeted mRNA inhibiting its translation. Assays are then performed to detect the RNAi activity.

How to make siRNA plasmids in sterile water?

Plasmid DNA (0.1-3.0 µg/µl in sterile water or TE Buffer, pH 8.0) Stealth RNAi or siRNA of interest (20 µM stock in 1X RNA Annealing/Dilution Buffer) or shRNA or miRNA expression plasmids of interest (0.1-3.0 µg/µl in sterile water or TE Buffer, pH 8.0)

How much siRNA is needed for Lipofectamine knockdown?

Tip: 20 µM dsRNA (i.e. siRNA or Stealth RNAi) = 20 pmol/µl. Note that for highly potent RNAi molecules (i.e. RNAi molecules inducing > 90% target knockdown), the amount of dsRNA or RNAi vector required to obtain effective knockdown may be less than the amounts specified in the table (Suggested Reagent Amounts and Volumes, above (see column 5).