Quantitative Analysis of caspase mRNA

To evaluate the expression of ca1pase, mRNA was extracted using a NucleoSpin Tri Prep kit (Macherey-Nagel) including DNase treatment. RNA concentration and quality were determined via a spectrometer (SpectraStar Nano, BMG Labtech). A subsample of 1 µg RNA was used for cDNA synthesis using the Precision nanoScript 2 Reverse Transcription kit (Primer Design) according to the manufacturer’s instructions. Reverse-transcription quantitative PCR (qPCR) was performed with the PrecisionPLUS qPCR Master Mix kit (Primer Design) containing cDNA (1:5 dilution) and the primer pair (Supplemental Table S1) in a Mx3005P qPCR system (Stratagene, Agilent Technologies). Melting curves were also completed. Primer efficiency was analyzed based on a cDNA dilution series with mean primer efficiency estimated using the linear phase of all individual reaction amplification curves and calculated according to Pfaffl (2001) (link). The succinate dehydrogenase (UniGene Cluster ID Ta.2218) and ADP-ribosylation factor (Ta.2291) genes were used as reference genes to normalize gene expression (Paolacci et al., 2009 (link); Evens et al., 2017 (link)). The normalized relative quantity (NRQ) of expression was calculated in relation to the cycle threshold (CT) values and the primer efficiency (E) of the target gene (X) and the reference genes (N), based on Rieu and Powers (2009) (link): NRQ = (EX)^−CT,X/(EN)^−CT,N.