add de Vries study and qpcr protocol

studies/deVries-2018/protocols/qRT-PCR.md

+## qRT-PCR
+
+qRT-PCR was performed using the miScript SYBR Green PCR (Qiagen, Hilden, Germany). miR482/2118 forward primers were designed based on the mature miR482/2118 sequences. miR482/2118 primer specificity was tested by creating a qRT-PCR product for each primer. These qRT-PCR products were purified, and each primer was tested with each qRT-PCR product to determine if and at what annealing temperatures the primers would bind to other miR482/2118 paralogues. For all miR482/2118 primers a binding-specific annealing temperature was determined (electronic supplementary material, table S1). The only exceptions were the primers for *Sl*miR482h and *Sp*miR482h, which annealed to miR482h as well as miR482 at all annealing temperatures. *Sa*miR482h was specific because of its slightly different mature miRNA sequence (electronic supplementary material, table S1). As a control, the expression of mature *Sl*miR156a/b/c, *Sl*miR166a/b, *Sl*miR168a/b and *Sl*miR172a/b was determined. miR390a was used as a reference due to its constant expression across treatments and time-points according to BestKeeper v.1 (Pfaffi et. al., 2004).
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+Expression of NBS-LRRs in *S. lycopersicum* was determined using the SsoAdvanced Universal SYBR Green Supermix (electronic supplementary material, table S1; Bio-Rad, California, USA). As reference genes, we used *SAND* (de Vries et. al., 2015), *TIP41* (de Vries et. al., 2015) and *Translation Initiation Factor 3 subunit H* (*TIF3H*; (de Vries et. al., 2017)).
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+Relative abundance and progression of *P. infestans* were measured using *Histone2a* (*PiH2a*). Expression of *PiH2a* at time-points 24 to 96 hpi was set relative to its expression at 24 hpi. The data were normalized with the plant reference genes (*SAND, TIP41* and *TIF3H*).
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+Relative expression was calculated according to [36]. Data were tested for normality using a Shapiro–Wilk test (Shapiro SS, Wilk MB., 1965) and equal variance using R v. 3.2.1. Comparisons between infections and mock control were tested using either a two-sample t-test or a Welch two-sample t-test for normally distributed data or a Mann–Whitney U-test (Mann HB, Whitney DR., 1947) for non-normally distributed data.
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