Quantitative RT-qPCR was used to determine the mRNA levels of the selected IRGs in the discovery cohort 1 of PPATs. Specifically, primers for the selected IRGs were used in combination with Brilliant III Ultra-Fast SYBR Green (ThermoFisher Scientific, Bedford, MA, USA) using the Stratagene Mx3000P system (Agilent Technologies, Madrid, Spain) following the manufacturer’s instructions. mRNA levels were determined to analyze the putative steady levels or the variability in the expression across the different groups of patients [i.e., with BPH or PCa and both under NW and OB conditions] using the MxPro 3.0 qPCR software (Agilent Technologies, Madrid, Spain).
Likewise, a microfluidic-based qPCR array (Standard BioTools, San Francisco, CA, USA) was used to simultaneously determine the gene expression levels of the 15 candidate IRGs in the second cohort of PPAT samples using a 48.48 Dynamic Array Plate (GE 48.48 Dynamic Array Reagent Kit with Control Line Fluid, Standard BioTools, San Francisco, CA, USA). Preamplification, exonuclease I treatment, and microfluidic-based qPCR array were implemented as previously described [41 (link),42 (link)] following the manufacturer’s instructions using the Biomark HD System and the Fluidigm Real-Time PCR Analysis v4.8.2 Software.
Likewise, a microfluidic-based qPCR array (Standard BioTools, San Francisco, CA, USA) was used to simultaneously determine the gene expression levels of the 15 candidate IRGs in the second cohort of PPAT samples using a 48.48 Dynamic Array Plate (GE 48.48 Dynamic Array Reagent Kit with Control Line Fluid, Standard BioTools, San Francisco, CA, USA). Preamplification, exonuclease I treatment, and microfluidic-based qPCR array were implemented as previously described [41 (link),42 (link)] following the manufacturer’s instructions using the Biomark HD System and the Fluidigm Real-Time PCR Analysis v4.8.2 Software.
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