results not shown) and the CT values of the biological triplicates. The negative inverted values were determined (i.e. 0.5 = 1/20.5 = 22.0) for fold change values smaller than one (,1). Genes were only regarded as upregulated with fold changes $2.0, and downregulated with fold changes #22.0, based on the same cut-off values used in a recent study [33]. SABioscienes quantitative PCR array The RNA extracted with the RT2 qPCR-Grade RNA Isolation Kit from liver and spleen collections of all mice at 72 hours post infection were diluted to 150 ng/ml in nuclease free water. Complementary DNA (cDNA) was prepared from the RNA using the RT2 First Strand kit (SABiosciences, Qiagen, U.S.A) as described by the manufacturer. A total of 1.2 mg of each RNA preparation was mixed with 56 genomic DNA Elimination buffer and the reaction incubated at 42uC for 5 minutes (total volume 10 ml). After the incubation, the reactions were immediately moved to ice and, subsequently, an equal volume of RT-coctail mix added (56 RT buffer, primers and external control mix, RT- enzyme mix and RNase-free water). These reactions were then incubated at 42uC for 15 minutes and 95uC for 5 minutes. The resultant cDNA of each preparation was then diluted 1:10 with nuclease-free water and stored at 220uC until tested. The 1:10 diluted cDNA was mixed with the master mix (26 SABiosciences RT2 qPCR Master Mix) and nuclease-free water, and aliquoted onto the PCR array plates containing primer sets (listed in Table S1)(25 ml of reaction mix per well)(PAMM-053, SABiosciences, Qiagen, Germany). Plates were run on an ABI 7500 cycler (Applied Biosystems, U.S.A). The following cycling program was used: 1695uC for 10 minutes, 40695uC for 15 seconds and 60uC for 1 minute, followed by the default melting curve program. Fluorescence was measured just after the 1 minute/60uC step. The cycle threshold (CT) values were determined using the cycler software and an automatic baseline adjustment (ABI 7500 Software Version 2.0.1, Applied Biosys- tems, U.S.A). Three mice per group were analyzed and average values calculated. Relative quantification data analysis (SABioscienes quantitative PCR array) Calculation of fold change values from the results of the SABiosciences quantitative PCR array relies essentially on the References 1. Woods CW, Karpati AM, Grein T, McCarthy N, Gaturuku P, et al. (2002) An outbreak of Rift Valley fever in Northeastern Kenya, 1997–98. Emerg Infect Dis 8: 138–144. 2. Swanepoel R, Coetzer JAW (2004) Rift Valley fever. In: Coetzer JAW, Tustin RC, eds. Infectious Diseases of Livestock. Cape Town: Oxford University Press. pp 1037–1070. 3. Rich KM, Wanyoike F (2010) An assessment of the regional and national socio- economic impacts of the 2007 Rift Valley fever outbreak in Kenya. Am J Trop Med Hyg 83: 52–57. 4. 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(1995) Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response. Journal of Medical Virology 46: 293–303. Protective Anti-RVFV NP Responses in Mice same principles as for the Quantitect qRT-PCR, making use of the 22DDCt method, except for the use of up to 5 housekeeping genes (Gusb, Hprt1, Hsp90ab1, Gapdh and Actb) for normalization of data. These calculations were done using the SABiosciences PCR Array Data Analysis Template Excel Utility (http://sabiosciences. com/pcrarraydataanalysis.php)(Qiagen, Germany). The negative inverted values were determined (i.e. 0.5 = 1/20.5 = 22.0) for fold change values smaller than one (,1). Genes were only regarded as upregulated with fold changes $2.0, and downregulated with fold changes #22.0. Statistical analysis The REST software uses a Pair-Wise Fixed Reallocation Randomization Test [106] with 2000 randomizations to determine a P-value which gives an indication of the statistical significance of fold changes. The SABiosciences PCR Array Data Analysis Template Excel Utility incorporates the calculation of a P-value using a T-test. Fold changes with P-values smaller or equal to 0.05 (#0.05) were taken as statistically significant. Three biological replicates were used at all experimental time points in all groups to enable statistical reliability of results. Supporting Information Table S1 Fold change in expression of 84 genes involved in activation of B- and T-cell immunity. Fold changes are shown for immunized mice versus non-immunized control mice (n = 3 per group) after RVFV challenge at 72 hours in liver, relative to expression in an age-related control group of mice (n = 3). (DOC) Acknowledgments We would like to thank Mrs. B. 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Gauliard N, Billecocq A, Flick R, Bouloy M (2006) Rift Valley fever virus noncoding regions of L, M and S segments regulate RNA synthesis. Virology 351: 170–179. 12. Lorenzo G, Martin-Folgar R, Rodriguez F, Brun A (2008) Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep. Vaccine 26: 5255–5262. 13. Liu L, Celma CC, Roy P (2008) Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins. Virol J 5: 82. 14. Lorenzo G, Martin-Folgar R, Hevia E, Boshra H, Brun A (2010) Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(2/2) mice induced by different DNA vaccination regimens. Vaccine 28: 2937–2944. 15. Lagerqvist N, Naslund J, Lundkvist A, Bouloy M, Ahlm C, et al. (2009) Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs. Virol J 6: 6. PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25027