7500 fast qpcr instrument

For quantitative PCR (qPCR) analysis, rosette leaves of two 23‐d‐old soil‐grown plants were pooled for each sample, with three biological replicates collected per sample. The analysis was performed as previously described (Major et al., 2020 (link)). Briefly, and according to manufacturer's instructions, RNA was extracted using the NucleoSpin plant RNA extraction kit (Macherey‐Nagel, Allentown, PA, USA). Following quality assessment by A260/A280 ratios, RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Waltham, MA, USA). qPCR reactions were run using the Power SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Fast qPCR instrument with a dissociation curve to confirm a single peak for each set of primers. Primer efficiencies for each primer pair were determined with LinRegPCR v.2012.0 and target gene expression was normalised to the expression of PROTEIN PHOSPHATASE 2A (PP2A).

Tissue segments were snap-frozen, then homogenized in ice-cold RLT buffer (Qiagen, United Kingdom) containing β-mercaptoethanol (1% v/v; Sigma) using a Precellys24 instrument. RNA was extracted using RNeasy mini-prep kits (Qiagen) and gene expression levels measured using a 1-step RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) master mix (Promega, United Kingdom) and a 7500 Fast qPCR instrument (Applied Biosystems, United Kingdom) using TaqMan probes (Qiagen) recognizing Ptgis (probe ID: Mm00447271_m1), Ptgs1 (probe ID: Mm00477214_m1), Ptgs2 (probe ID: Mm00478374_m1), or the housekeeping genes 18S (probe ID: Mm03928990_g1) and Gapdh (probe ID: Mm99999915_g1). Data were analyzed by the comparative threshold method, with relative expression levels normalized to those to 18S and Gapdh and experimental control groups.