Table 2.

A simulated chart for checking qPCR data. For simplicity, a sample number code can be used on tubes and extraction bags with a spreadsheet containing the precise details. Technical duplicates of qPCR reactions for the specimen are run and the Cq determined by the qPCR software. If the technical duplicates are close to one another, similar to those in Table 2, then one can proceed to qPCR singlet reactions. The reference gene RPS5a was run and also the antimicrobial peptide (AMP) gene Hymenoptaecin (“Hym”). The ΔCq = Cq _RPS5aminus Cq _{gene of interest “Hym”}) was calculated. We observe that the honey bees that were inoculated with Nosema followed by natural product dosing (+Nosema+NP) had a higher relative gene expression of hymenoptaecin than the control treatment of a Nosema infection without the natural product dosing (+Nosema-NP). qPCR data should be coupled with survival data. CoV: Coefficient of Variation; 1st: first of two technical qPCR reaction replicates; 2nd: second of two technical qPCR reaction replicates; Replicate difference: the Cq difference between each technical qPCR reaction; Average: the average of the two technical qPCR reaction runs (not done when run in singlets); ΔCq: the difference between the reference gene (Rps5a) and the target gene (e.g., Hym), whereby a higher number represents higher relative gene expression.

Sample ID#	Treatment	Replicate	Rps5a 1st	Rps5a 2nd	Average	Replicate Difference	CoV
1	+Nosema+NP	1	20.19	19.75	19.97	0.44	1.557971876
2	+Nosema-NP	1	21.1	20.03	20.565	1.07	3.67908707
Sample ID#	Treatment		Hym 1st	Hym 2nd	Average	Replicate Difference	CoV	ΔCq
1	+Nosema+NP	1	25.2	26.1	25.65	−0.9	2.481076425	−5.68
2	+Nosema-NP	1	28.2	28.9	28.55	−0.7	1.733711898	−7.985