Twenty-four hours after the transfection, the cells were detached, filtered, and then analyzed with a BD FACSAria II (BD Biosciences). The fluorescence of hmAG1, hmKO2, tagBFP, and hdKRed was measured using a blue laser with a FITC filter, a green laser with a PE filter, a violet laser with a Pacific Blue filter, and a violet laser with a Qdot 605 filter, respectively. From the analyzed cell populations (10,000 counts), dead cells and debris were gated out according to forward scatter and side scatter, and noise in the hmKO2 measurement was removed on the basis of the height and area of the signal intensity. Using the fluorescence intensities of a cell, the expression from the synthetic mRNAs was evaluated with the indices defined in this study: fluorescence ratio, reporter expression, and relative expression (see Supplementary Text for details). In the experiments using a set of four reporter mRNAs, the fluorescence intensities were compensated to determine the levels of the four fluorescent proteins, as described previously (38). Briefly, a spillover matrix of four fluorescent proteins into the four detection channels was experimentally determined for each transfection series on each cell type. The values in the matrix were obtained by fitting flow cytometric data from the cells transfected with either one of the four reporter mRNAs (control mRNAs) or untransfected cells with gnuplot (www.gnuplot.info). The fluorescence intensities in single cells were compensated with the compensation matrix, which is the inverse of the spillover matrix, and then used to calculate the fluorescence ratios.

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