**3.6. Statistical analysis of data**

QIAzol lysis reagent for 10 min at 30 Hz in a Tissuelyzer LT (Qiagen, UK). Lysates were mixed with 100 μL chloroform, transferred to pegGold PhaseTrap tubes (PeqLab, UK) and centrifuged for 5 mins at room temperature. The aqueous phase was poured into fresh tubes, mixed with 1.5 volumes of ethanol and applied to Qiagen RNeasy columns (Qiagen, UK). RNA was purified according to the manufacturer's instructions (Qiagen, UK). RNA integrity was assessed using an Agilent Bioanalyzer and RIN was >8 for all samples. Purity and quantity were measured using a NanoDrop spectrophotometer; for all samples the absorbance peak was at 260 nm, A260/280 > 2 and A260/230 > 1. About 800 ng of RNA were reverse transcribed using a Quantitect reverse transcription kit (Qiagen, UK) in a 10 μL reaction according to the manufacturer's instructions. This RT kit includes a mandatory gDNA wipe out step. The

Two microlitres of cDNA were amplified in a 10 μL reaction using Agilent Brilliant III SYBR Ultra-Fast SYBR Green mix with each primer at a final concentration of 500 nmol/L. The notemplate control reaction contained 2 μL of tRNA (0.5 μg/mL). DNA standards (10^7–10^1 copies/rxn) for each gene were included in each run. Reactions were pipetted robotically using a Qiagility (Qiagen, UK). Amplification parameters were: 95°C for 3 min followed by 40 cycles of 95°C for 5 sec, 57°C for 1 sec in a Rotor-Gene 6000. Melt curves were checked

**Figure 2.** The effect of dietary plant extracts (PE) on the normalised mRNA copy number (per reaction) of (a) CD40 LG,

(b) IL-12B, (c) INFG, (d) IL-6 in chicken caecal tonsils. Error bars represent ±1 pooled SEM.

completed reaction was diluted 10-fold with 5 μg/mL tRNA in water.

110 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

*3.5.2. Quantitative real-time PCR*

Data were statistically analysed by two way analysis of variance (ANOVA) using a 2 × 2 factorial arrangement of treatments, blocked by experiment. The main effects were the cereals (maize and wheat) and additives (with and without PE). All data were processed using the procedure of Genstat (18th Edition) statistical software (IACR, Rothamstead, Hertfordshire, UK). In all instances, differences were reported as significant at P < 0.05. Graphics (**Figure 2**) were produced in "ggplot2" package version 2.2.1. [41] using R version 3.4.1. [42].
