Systematic analysis of bicistronic reporter assay data - PubMed (original) (raw)

Systematic analysis of bicistronic reporter assay data

Jonathan L Jacobs et al. Nucleic Acids Res. 2004.

Abstract

Bicistronic reporter assay systems have become a mainstay of molecular biology. While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties. In general, a primary dataset measures the paired expression of two internally controlled reporter genes. The expression ratio of these two genes is then normalized to an external control reporter. The end result is a 'ratio of ratios' that is inherently sensitive to propagation of the error contributed by each of the respective numerical components. The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects. A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems. In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems. Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed -1 frameshift signals. These frameshift signals originate from the L-A virus, the SARS-associated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes. Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed -1 frameshifting are statistically significant. A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website (http://dinmanlab.umd.edu/statistics). These spreadsheets could be easily adapted to any bicistronic reporter assay system.

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Figures

Figure 1

Figure 1

Visualization of data from control reporter _C_1 and experimental frameshift reporters _F_1, _F_2 and _F_3. The raw luminescence values for _C_1, _F_1 and _F_2 are linear and the ratio values of firefly/Renilla are normally distributed. The data for _F_3, however, are neither linear nor normally distributed. (A–D) RLUs of firefly and Renilla expressed from control reporter _C_1 and experimental frameshift reporters _F_1, _F_2 and _F_3, respectively. Outliers are shown by solid data points. (E–H) Normal probability plots for data from _C_1, _F_1, _F_2 and _F_3, respectively. The _x_-axis corresponds to the expected _z_-score of each data point. The _y_-axis is the ratio of firefly to Renilla RLU values. The trend line shown is based on the linear regression of the data and represents the expected firefly/Renilla RLU ratio for a given _z_-score. Normal PPCCs for these (E–H) are shown in Table 1.

Figure 2

Figure 2

Visualization of data from control reporter _C_2 and experimental frameshift reporter _F_4 under the effects of 20 μg/ml of anisomycin, an inhibitor of −1 PRF (26). The raw luminescence values are linear and the ratio values of firefly/Renilla are normally distributed. (A–D) RLUs of firefly and Renilla expressed from each reporter. Outliers are shown by solid data points. (E–H) Normal probability plots for each reporter. (A and E) _C_2, no drug. (B and F) _C_2 with anisomycin. (C and G) _F_4, no drug. (D and H) _F_4, with anisomycin. See Table 1 for details.

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References

    1. Coleman H.M., Brierley,I. and Stevenson,P.G. (2003) An internal ribosome entry site directs translation of the murine gammaherpesvirus 68 MK3 open reading frame. J. Virol., 77, 13093–13105. - PMC - PubMed
    1. Venkatesan A., Sharma,R. and Dasgupta,A. (2003) Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells. Virus Res., 94, 85–95. - PubMed
    1. Imbert I., Dimitrova,M., Kien,F., Kieny,M.P. and Schuster,C. (2003) Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3′ NTR even in the presence of viral structural or non-structural proteins. J. Gen. Virol., 84, 1549–1557. - PubMed
    1. Meskauskas A., Harger,J.W., Jacobs,K.L. and Dinman,J.D. (2003) Decreased peptidyltransferase activity correlates with increased programmed −1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae. RNA, 9, 982–992. - PMC - PubMed
    1. Novac O., Guenier,A.S. and Pelletier,J. (2004) Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res., 32, 902–915. - PMC - PubMed

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