antibodies were converted to percentage positivity (PP) values by the following calculation: PP = Mean OD sample/Mean OD positive control 6100, using the OD values described above for IgG, IgG1 and IgG2A to calculate PP values for those specific subclasses. The PP values determined for each of the three biological replicates at each time point (three mice sampled per time point) were then used to calculate the biological mean PP values and standard deviations (S.D.) at each sampling point for the different IgG subclasses. RVFV challenge During the RVFV challenge phase of the experiment the mice were housed as during the immunization part except for being moved to a Biosafety level 3 animal facility. Vero cells were cultivated in Eagles Minimal Essential Medium (EMEM) (BioWhitaker, MD, U.S.A) containing L-Glutamine, non-essential amino acids, antibiotics (100 IU penicillin, 100 mg streptomycin and 0.25 mg amphotericin B) and 10% foetal bovine serum (Gibco) and maintained at 37uC in 5% CO2 incubator. The SPU22/118 KEN 07 strain of RVFV was isolated from a RVF human case during the 2007 Kenyan epidemic [104]. Second passage of the virus, propagated in Vero cells, was used for the challenge of mice on day 28 after the booster immunization. The recNP immunized, adjuvant control and PBS control mice were inoculated s.c. with 100 ml of 107.0 TCID50/ml RVFV, whereas the age related control group of mice were ‘‘mock’’ challenged with 100 ml uninfected Vero cell culture supernatant. Mice were monitored twice daily for clinical signs. Three mice were randomly collected from each group, euthanized by CO2 asphyxiation and liver and spleen tissues collected at the following time points after RVFV challenge: 3, 6, 12, 24, 72 and 120 hours. A piece of approximately 50 mg of each tissue was collected into an RNA stabilization reagent (RNAlater, Qiagen, Germany) according to the instructions of the manufac- turer, and stored at 270uC until further processing. The remaining tissue was stored in cryotubes at 270uC for viral load determination. Virus titrations Liver and spleen tissues were homogenized as 10% (w/v) suspensions in EMEM containing L-glutamine, non-essential amino acids and antibiotics (100 IU penicillin, 100 mg streptomy- cin and 0.25 mg amphotericin B) using a Tissuelyser II and 5 mm stainless steel beads (Qiagen, Germany) according to the manufacturer’s instructions (4 min, 25 Hz). After centrifugation at 10,0006 g for 3 minutes the supernatants were collected and stored at 270uC until tested. Titrations were performed as described previously [17]. Virus titers, calculated by the Ka¨rber method [105], were expressed as median tissue culture infectious dose (TCID50) per gram of tissue. Means and standard deviations from the means were determined based on three animals per group per each time point tested. RNA extraction The tissues were moved from RNAlater into lysis buffer (Buffer RLT, Qiagen, Germany) and lysis-homogenization performed by using a Tissuelyser II as described above. Lysates were centrifuged for 3 minutes at 13,200 rpm at room temperature and superna- tants collected. Extraction of RNA from the supernatants was performed by using the RNeasy Mini Kit (Qiagen, Germany). A 50% ethanol was used to increase RNA yield from livers and 70% ethanol for spleens as suggested by the manufacturer, followed by on-column Dnase digestion using the Rnase-free Dnase set (Qiagen, Germany), to remove genomic DNA. The RNA was Protective Anti-RVFV NP Responses in Mice eluted in Rnase-free water, the concentration determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, U.S.A) and stored at 270uC until further testing. For testing with the SABiosciences quantitative PCR array (as described below), further RNA cleanup was performed on RNA already extracted from livers and spleens collected at 72 hours post infection, as per the manufacturer’s instructions. This cleanup, which introduces an additional Dnase digestion, was performed using the RT2 qPCR-Grade RNA Isolation Kit (SABiosciences, Qiagen, U.S.A) as per the manufacturer’s instructions. The RNA was eluted in Rnase-free water, the concentration determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scien- tific, U.S.A) and stored at 270uC until further testing. Quantitect qRT-PCR The RNA extracted from all experimental mice at all time points after RVFV challenge were diluted to 10 ng/ml and used in the qRT-PCR reactions. The Quantitect qRT-PCR reactions were performed as described by the manufacturer (Quantifast SYBR Green RT-PCR kit, Qiagen, Germany) using a Lightcycler 1.5 (Roche, Germany). Briefly, a reaction mix was prepared by mixing 26 Quantifast SYBR Green RT-PCR Master Mix (HotStarTaq Plus DNA Polymerase, Quantifast SYBR Green RT-PCR buffer, dNTP mix and ROX passive reference dye), 106 Quantitect Primer set (for detection of the genes for Gapdh, Il10, Ifng and Ifnb1), Quantifast RT-Mix (Omniscript RT and Sensiscript RT), template RNA (10 ng) and RNase-free water to a final volume of 20 ml per reaction. This mix was transferred to 20 ml Lightcycler Capillaries (Roche, Germany) and run on the Lightcycler 1.5 using the following cycles: 16 reverse transcription (10 min, 50uC), 16 hotstart PCR activation (5 min, 95uC) and 406 cycles of denaturation (10 sec, 95uC) and annealing/ extension (30 sec, 60uC), with fluorescence data collection just after the annealing/extension step. The threshold cycle (CT) values were determined using the second derivative maximum method (Lightcycler Data Analysis Software version 3.5.28, Roche). The CT values were then used for relative quantification (as described below). RNA was extracted from the liver and spleen of a normal BALB/cOlaHsd mouse to generate standard curves in order to determine PCR reaction efficiencies (necessary for the relative quantification calculations) with primer sets for different genes (GAPDH, IL-10, IFNa˜ and IFNaˆ1). Dilution series were prepared and the following amounts of RNA tested in duplicate using all primer sets: 30 ng; 15 ng, 7.5 ng, 3.75 ng and 1.875 ng. The CT values and their corresponding template amount values were then used to determine the PCR reaction efficiencies using the Relative Expression Software Tool (REST, Qiagen, Germany) [106] as described by the manufacturer (results not shown). Relative quantification data analysis (Quantitect qRT-PCR) Threshold values (CT) from biological triplicates for the different genes analyzed as determined for the immunized mice, adjuvant control and PBS control mice were first normalized to the CT values of the housekeeping gene (Gapdh) analyzed in the same samples, and further normalized to the CT values from the non- infected control mice to determine the relative changes in gene expression compared to age related normal mice. This is the so- called 22DDCt and the result is a fold change value that is an indication of the expression level of a gene in the experimental group being higher or lower than the expression level in the normal age-related control group [107]. These calculations were done using the REST software (Qiagen, Germany) which uses the PCR efficiencies for different primer sets (as determined above, PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e25027