Real time pcr analysis software

The C1 Single-Cell Auto Prep Array for PreAmp (Fluidigm) was used for harvest of RNA, cDNA synthesis, and preamplification of cDNA (18 cycles of PCR for the target genes) from single cells according to the manufacturer’s instructions. After loading cells onto an integrated fluidic circuit, we checked all 96 chambers by microscope to verify capture of a single cell. Thereafter, preamplified cDNA was harvested and subjected to qPCR. The Biomark HD system (Fluidigm) combined with EvaGreen chemistry (Bio-Rad) was used for the qPCR assay. To increase specificity, we used nested primers (one pair for the preamplification step and another pair for the subsequent qPCR of 30 cycles). The sequences of the primers used in this study are indicated in Table S3. Raw data were processed by Fluidigm Real-Time PCR analysis software, and the melting curve was used to determine the pass/failure call of qPCR. Data were analyzed with the R program using the Singular Analysis Toolset package (Fluidigm). Any chamber that contained more than one cell was excluded from analysis; outlier cells that had low global expression were also excluded. The level of detection value was set to 24 cycles according to the manufacturer’s recommendation.

The raw data of the protein profiling were first normalized following the standard protocol from the manufacturer and using the Olink Wizard of GenEx software (MultiD, Göteborg, Sweden). For each data point, the raw quantification cycle value (Cq-value, in log2 scale) was exported from the Fluidigm Real-Time PCR Analysis Software. The Cq-value is defined as the calculated cycle number at which the PCR product crosses a threshold of detection and is used to represent the expression levels of respective proteins in the current study. The first step of normalization was to subtract the raw Cq-value for the extension control for the corresponding sample to correct for technical variation. The calculated Cq-values (dCq-value) were further normalized against the negative control determined in the measurement, which yielded ddCq-values (hereafter, Cq-value, in log2 scale) and could be used for further analyses. Limit of detection (LOD) was defined as the mean value of the three negative controls plus three calculated standard deviations. A total of 30 samples with invalid test results were excluded from this analysis. Missing data and data with a value lower than LOD were replaced with LOD in the following statistical analyses.

The BioMark^TM real-time PCR system (Fluidigm, USA) was used for high-throughput microfluidic real-time PCR amplification using the 48.48 dynamic arrays (Fluidigm) as described [14] (link). In short, amplifications were performed using 6-carboxyfluorescein (FAM)- and black hole quencher (BHQ1)-labeled TaqMan probes with TaqMan Gene expression master mix (Applied Biosystems, France). The thermal profile comprised 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of a 2-step amplification profile consisting of 15 s at 95 °C for denaturation and 1 min at 60 °C for annealing and extension. Data were acquired on the BioMark^TM Real-Time PCR System and analyzed using the Fluidigm Real-time PCR Analysis software to obtain cross point (Cp) values. Negative controls with water were included per chip. The detection of D. reticulatus DNA served as a confirmation of the tick species tested and as a positive control of the DNA extraction. A positive processing control, which is a DNA extract from the EDL933 strain of Escherichia coli, was added to each sample.