Quantstudio design analysis software

Total RNA was extracted by PureLink RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. Quantitative real-time PCR of cDNA was performed by the PrimeScript RT Master Mix (TaKaRa, China). To examine expression of TeLCYB, cDNA sample was used as the template in quantitative real-time PCR assay by Powerup SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA). The 16SRNA was used as the internal standard. The primers of targeted mRNA are listed in Table S2. Raw data of RT-PCR were investigated using Quant Studio Design & Analysis Software (Thermo Fisher Scientific). Relative expression levels were analyzed by the 2^−ΔΔCT method.

Whole genomic DNA was isolated from blood using QIAamp DNA Blood Mini Kit (Qiagen Iberia, Madrid, Spain). Intermediate HLA genotyping resolution (four digits, the corresponding “P” for every 2^nd field allele typing was discarded to clarify the text) was performed with 2μg of genomic DNA using LinkSēq™ HLA ABCDRDQB1 384 kit (One Lambda, Thermo Fisher Scientific España, Madrid, Spain), which allows testing human class I classical HLA-A, -B, -C alleles and class II–DR, -DQ and–DP alleles, including Bw4 and Bw6 genotypes. qPCR was performed using QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), according to manufacturer’s instructions and using the following conditions: 95°C, 2 min; 36 cycles: 95°, 15 seconds; 64°C, 1 min. A dissociation profile was added using the following conditions: 95°C, 15 seconds; 65°C, 30 seconds. The dissociation data corresponding to the melt curve were collected per well between 65°C and 95°C using QuantStudio™ Design & Analysis Software (Thermo Fisher Scientific). These data were exported to SureTyper software (One Lambda) for the interpretation and identification of HLA alleles.

Total RNA was isolated with Ribozol (VWR) and 1 µg of RNA was reverse transcribed using the High-Capacity cDNA reverse transcription kit (Life Technologies). qPCR was performed using PowerUp SYBR Green master mix and analyzed with the QuantStudio design & analysis software (Thermo Fisher). qPCR primers used in this study are listed in the Supplementary Table 1. Relative gene expression levels were calculated using a relative‐standard curve method. Each gene expression levels were normalized by the expression levels of Gapdh except in Figure 9I and 9 M, where by β-actin was used for normalization.

To evaluate the analytical specificity and sensitivity, the threshold in qPCR was determined automatically, as well as, Ct values, standard curve plotting, coefficient of determination (R²) and efficiency (E = 10^−1/slope) of each PCR run were analysed using QuantStudio Design & Analysis software (ThermoFisher Scientific, Life Technologies, Carlsbad, CA, USA). The LOD of the qPCR assay was determined as the amount of S. parasitica DNA, for which over 95% of positive samples were detected [22 (link)]. Variation in qPCR assay results between replicate samples was evaluated from the Ct mean and ±SD of the replicates.
The correlation between the quantity of S. parasitica DNA measured by qPCR (ng/L) and the Saprolegnia sp. spore number (spores/L) measured with the MWP method was assessed using Pearson’s correlation coefficient with 95% confidence interval (CI) using IBM SPSS Statistic software (version 1.0.0.1447). The respective repeatability was estimated using normalized values and interclass-correlation coefficient (ICC) [23 (link)]. Water sample result from tank 1a was excluded from these analyses, as the amount of Saprolegnia in the water sample was above the detection limit of the MWP method.