Ariamx real time pcr system

Phytoplasma titer was assessed by qPCR. The host-to-phytoplasma DNA ratio was determined using the AriaMx Real-Time PCR System, which utilized Brilliant SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA). The 16S rRNA gene fragment from PPT phytoplasma DNA and tomato actin (internal control) fragment from the total DNAs were amplified using MPPLPPT16SF2/MPPLPPT16SR2 (Supplementary Table S1, [30 (link)]) as well as Sl-ACT-F/Sl-ACT-R primers, respectively (Supplementary Table S1). To determine the fold changes in phytoplasma titer, the expression levels were normalized to the internal control. A minimum of three independent biological samples were used. The following thermocycling conditions for qPCR operation were used: an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of amplification. Each cycle included denaturation at 95 °C for 5 s, annealing, and extension at 60 °C for 10 s. Statistical analysis was conducted using a Student’s t test in Microsoft Excel (Microsoft Corporation, Seattle, WA, USA) to assess the significance of differences between groups.

The slices used for gene expression analysis were collected and processed with Allprotect tissue reagent (Qiagen, Manchester, UK) as per the manufacturer’s instruction. The RNA extraction was performed using an RNeasy Mini kit (Qiagen, Manchester, UK). To generate cDNA, 200 ng of RNA was used with the First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Oxford, UK) according to the manufacturer’s protocol. Each sample was diluted 1:20 with RNAse-free water. Polymerase chain reaction was performed using SYBR green (ThermoFisher Scientific, Oxford, UK) in a 10 μL real-time PCR reaction. The following PCR conditions were used for all experiments: denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, then at 60 °C for 60 s. All primers were designed on Primer Express v3.0 and are available in Supplementary Table S1. Reactions were duplicated for each sample and quantified in the AriaMx Real-time PCR System (Agilent, Stockport, UK). Data were expressed as arbitrary units, and the relative expression of target genes (FASN, SCD1, HMGCR, ELOVL6) was corrected to the geometric average of two housekeeping genes (determined using BestKeeper tool, BK): 18S, GADPH. Fold change across conditions was calculated by dividing the relative expression value of each gene by the geometric mean relative expression value from the control samples.

Both total bacteria and load of O. oeni were quantified by qPCR reactions based on detection of SYBR fluorescence. The qPCR reactions were performed using an AriaMx real-time PCR System (Agilent Technologies), using primers and programs described in Table 1. PCR amplification was performed in 10 μL of mix containing 1 μL of DNA 0.5 pmol/mL of each respective primer 5 μL of LightCycler 480 SYBR Green I Master (Roche) and 3.5 μL of Milli-Q sterile H₂O. All samples were analyzed in triplicate. The statistical analyses among bacterial loads valleys were determined by ANOVA using R Development Core Team (2008 ), and the post-hoc test was performed by pairwise.t.test.

Female medaka of esr2a^+/− and esr2a^−/− (age: three to four months) were deeply anesthetized and their pituitaries were collected for qRT-PCR.
Total RNA was extracted from the pituitaries using the NucleoSpin RNA plus (TaKaRa, Kusatsu, Japan) according to the manufacture’s instruction. Total RNA samples were then reverse transcribed by PrimeScript^TM RT Master Mix (TaKaRa). For qRT-PCR, the cDNA was amplified by KAPA SYBR FAST qPCR kit (Nippon Genetics, Tokyo, Japan) with AriaMX Realtime PCR System (Agilent Technologies, Santa Clara, CA). The temperature profile of the reaction was 95 °C for 3 minutes, 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 10 seconds. The PCR products were verified using melting curve analysis. The data were normalized by a housekeeping gene, ribosomal protein s13 (rps13). The melting curve analyses were conducted to ensure that the amplicons were the same as the sequence-certified ones. The primer pairs used in the real-time PCR are listed in the primer list (Supplementary Table 1).