Discrepancies in qPCR-based gene quantification and their dependencies on soil properties, inhibitor presence, and...

The complexity and heterogeneity of soil samples necessitate the inclusion of extensive purification steps prior to genomic assays, such as quantitative PCR (qPCR). Although conventional DNA extraction kits have notably enhanced the convenience of the process, those designed for soil vary considerably in terms of reagents, time, and equipment. Therefore, purified gDNA quality varies depending on the DNA extraction kits used, which leads to discrepancies in gene quantification using qPCR. This issue can be amplified considerably when more complicated (or contaminated) soils are analyzed, even if extensive DNA extraction is employed. Here, we evaluated the influences of the DNA extraction method to the gene quantification using qPCR across soil types. Further Mg²⁺ ion spiking experiments were performed to observe multiple inhibitory effects on qPCR analysis performance. The results suggest that discrepancies in gene quantification are evident in the presence of qPCR inhibitors in soil samples. Furthermore, discrepancies in quantification results are exacerbated by gDNA template quality, which is attributed to DNA extraction. The observed multiple inhibitory effects underscore the importance of careful consideration of both DNA template quality and soil type to ensure more accurate gene quantification in soils.