Our data suggest that the corresponding pre-miRNA coding sequence is detected by the stem-loop primer. There are data that reverse transcriptases can use DNA as a template, therefore this DNA dependent DNA polymerase activity might be an explanation for our observation. However, the extent of the DNA-derived false Dyclonine HCl detection varied among different miRNA targeting assays, and plasmid DNAs had more pronounced effects on the detection than gDNA contaminations. The significance of DNA contamination is further underlined by the fact that miRNA expression studies are often carried out on transiently transfected cells, which contain a significant amount of plasmid DNA originating from the used expression vector. Additionally to this, DNA and RNA Desogestrel molecules are both detected at 260 nm by spectrophotometry, therefore DNA contamination also disturbs the accurate measurement of RNA concentration. All these factors imply that extensive DNase treatment is a critical part of this miRNA quantification protocol which cannot be omitted when using certain RNA isolation methods. Our data show, that in contrast to total RNA isolation using either Trizol reagent or mirVana Kit, no significant DNA contamination present in the RNA samples when applying small RNA isolation by the mirVana Kit. In addition to the technical issues described above, the recently discovered isomiR species impose another challenge on miRNA detection by the stem-loop qPCR technique, as the sequence diversity of miRNA species could be quite extensive both at the 59 and the 39 ends. Although there are emerging data on the existence of this variability, neither all mechanisms responsible for the generation of isomiRs nor their potential functional differences are clear as yet. Even if 39 variability appears to be redundant in function at present, it represents a problematic issue not only for stem-loop qRT-PCR, but also for miRNA detection by the poly-tailing based methodology. Therefore, the a priori knowledge of the exact 39 sequence is a prerequisite for designing an accurate, specific assay for any particular small RNA species, and examining miRNA databases and available online deep sequencing data is strongly recommended.