Kapa sybr fast qpcr reagent

Cultures were grown at 30° with constant shaking for 12 hr (in the absence of Cm) or 14 hr (in the presence of Cm). RNA was isolated using the QIAGEN (Ontario, Canada) RNeasy Plant Mini Kit. RNA integrity was measured using an Agilent 2100 Bioanalyzer system and the Agilent RNA 6000 Nano kit (Agilent Technologies). Only those samples that had an RNA integrity number value of >9 were used for cDNA synthesis. First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). Reactions were performed using KAPA SYBR Fast qPCR reagents (KAPA Biosystems) and the StepOnePlus qPCR system (Applied Biosystems, Foster City, CA). Data were analyzed using StepOne software (Applied Biosystems). The β-tubulin gene was the internal control for ΔΔCt quantification. The qPCR data for each gene examined were obtained from four biological replicates, each with three technical replicates.

For qPCR, cells were grown to confluence in 35mm dishes, synchronized by temperature cycles and changed in to serum and B27-free “Air Media” as described above. After treatment for the time indicated, cells were harvested and RNA was extracted using the RNeasy mini kit (QIAGEN) using the recommended protocol. On column DNase digestion was performed. cDNA was generated from these samples using the iScript cDNA synthesis kit (BioRad) using a total RNA concentration of 100 ng per sample. Samples were then kept on ice while they were made up to a volume of 100 μL with nuclease-free water. Small quantities of all samples were pooled and subsequently serially diluted to make standards. Samples were diluted ten-fold to ensure their values fell within the standard curve. qPCR was carried out using a Prime Pro 48 machine (Techne) and KAPA SYBR Fast qPCR reagents (KAPA Biosystems) as per manufacturers instructions. The primer sequences used are described in the key resources table. All pairs of primers were annealed at 58°C, and a melt curve performed. Data was then analyzed and outliers excluded using the Prime Pro Study software. Values were normalized to the standard curve and against RNS18.

The Epichloë biomass in pseudostem tissues was quantified by using a quantitative PCR (qPCR) method (Cook et al., 2009 (link)). Epichloë biomass is expressed as picograms of Epichloë DNA per nanogram of total (plant and Epichloë) DNA (i.e., pg/ng total DNA). The quantification of Epichloë DNA is based on a 153‐bp DNA fragment amplified from the nonribosomal peptide synthetase gene NRPS1 (Rasmussen et al., 2007 (link)). For the quantification of the fungal endophyte, a standard curve (20, 10, 1, 0.1, 0.01, and 0.001 ng fungal DNA) was prepared from DNA extracted from a pure culture of the Fl1 WT strain extracted using the ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research Corp.). Three pseudostem tissues (1 cm above roots) from a single plant were pooled and at least four biological replicates were assayed on the plants infected with each construct. Total DNA was extracted from the pseudostem tissues using the NucleoSpin Plant II genomic DNA extraction kit (Macherey‐Nagel GmbH) and quantified using a Qubit fluorometer with the Quant‐iT dsDNA HS Assay kit (Invitrogen). qPCR was performed using KAPA SYBR FAST qPCR reagents (KAPA Biosystems) in a 10‐μL reaction containing total DNA (10 to 100 ng) and 200 nM each of the primers NRPS1_ qPCR_F and NRPS1_ qPCR_R in a Light‐Cycler 480 (Roche Applied Science).
A list of primers used in this study can be found in Table S2.