Guidelines for transfection

Guidelines for transfection of RNA

The transfection of RNA has become extremely important with the discovery of RNA interference (RNAi)-mediated gene silencing using short interfering RNA (siRNA).

Various forms of RNA, such as mRNA, in vitro transcribed RNA, viral RNA, RNA oligos, siRNA, and ribozymes, can be used for transfection. If using mRNA, the presence or absence of features such as a 5' cap, internal ribosomal entry site, or poly-A tail can have a significant effect on efficiency of transfection.

Optimal transfection results are achieved when RNA of the highest purity, free of contaminating DNA and proteins, is used for transfection. It should be considered that if the expressed gene product is toxic to the cell, overexpression may lead to cell death.

For RNAi experiments, high-purity, ready-to-use siRNA is available from several commercial suppliers. It is essential to use the correct sequence to achieve efficient silencing. Note that silencing the expression of essential genes may lead to cell death. For more information, see Guidelines for transfection of siRNA.

No currently available purification method can guarantee that RNA is completely free of DNA, even when no DNA is visible on an agarose gel. For RNA transfection, treatment of the purified RNA with RNase-free DNase or comparable methods is recommended.

Ribonucleases (RNases) are very stable and active enzymes that do not generally require cofactors to function. Since RNases are difficult to inactivate and minute amounts are sufficient to destroy RNA, plasticware, glassware, or solutions should only be used after eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases during the transfection procedure. In order to create and maintain an RNase-free environment when working with RNA, proper microbiological, aseptic technique should be followed, and the use of sterile, disposable plastic tubes is recommended.

The following factors must be optimized for the best results in RNA transfection:

  • Cell density at the time of transfection
  • The ratio of RNA to transfection reagent
  • The time of incubation with the transfection complexes

We recommend careful optimization of these parameters for every cell type and RNA combination used. Once optimized, these parameters should be kept constant in all future experiments with each particular combination.

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