Still, interpretations of combined quantitative measurements and amplicon sequencing data should consider basic differences between the combined methods.
For example, Zhang and colleagues applied 16S rRNA gene amplicon sequencing together with qPCR and cell-counting approaches, and found that qPCR as a cell enumeration method correlates with other forms of cell quantification \cite{Zhang2017}. This and other studies support the use of multiple approaches to improve the inferences that can be made from microbial community studies, particularly when these studies are conducted in heterogeneous environments or compare across different sampling sites or time periods.
However, the ability of qPCR to provide a quantitative measure for microbial abundance in the soil remains limited by technical challenges and the properties of soil samples. Quantification through this approach requires an amplified external standard for each assay, the proper quantification of which is critical for reliable results. Additionally, the sensitivity of the qPCR approach to differences in gene counts between samples depends on the total amplification efficiency of the assay, as well as the difference in amplification efficiency between the sample (a mixed community) and the standard (typically stemming from a single species). The senstivity and accuracy of qPCR is also limited by the presence of impurities, such as humic substances, that are commonly present in nucleic acid extractions from soils and are known to inhibit in-vitro enzymatic reactions such as PCR. The presence of such impurities leads to underestimation of gene or transcript copy numbers. In samples with very high nucleic acid purity, qPCR can reliably detect around twofold difference in copy numbers between samples and down to 100 copies \cite{Taylor_2019}. However, for most environmental samples the thresholds are often much higher. Reproducibility is also of concern in qPCR as identical assays can produce significantly different results when performed by different operators, qPCR cyclers, or using different kits \cite{Ebentier2013}.