IRES will not be efficiently translated due to a lack of factors necessary for translation initiation at an IRES in these cells.

RNA quality
Optimal transfection results are achieved when RNA of the highest purity is used for transfection. Therefore only RNA of the highest quality, which is free of contaminating DNA and proteins, should be used. RNA purified with RNeasy Kits and Oligotex mRNA Kits is highly recommended for transfection. We strongly recommend checking RNA quality before starting the transfection. RNA concentration and purity can be determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) using a spectrophotometer. An absorbance of 1 unit at 260 nm corresponds to ~40 g RNA per milliliter. RNA integrity and size can be checked by denaturing agarose-gel electrophoresis and ethidium bromide staining. The RNA should appear as a sharp band on the stained gel. If the band is not sharp but appears as a smear containing smaller sized RNAs, it is likely that the RNA has suffered major degradation during its preparation. The functionality of the transcript can be determined by in vitro translation.

Handling RNA
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, do not use any plasticware, glassware, or solutions without first 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, the precautions listed below must be
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