For RNA samples, as it was expected, both miR-1226-3p and miR-33b-5p probes resulted in significantly higher detected mature miRNA levels from the corresponding miRNA Hesperidin overexpressing cell line than in the controls. Concerning plasmid DNAs, apparently similar amount of miR-33b-5p was detected from mir33b encoding plasmid as from mir-33b overexpressing cell line derived RNA. This striking false effect was even more pronounced in the case of miR-1226-3p when the mir-1226 expression plasmid served as a template. There was 9 Ct difference compared to the mir-1226 overexpressing cell line derived RNA sample, and about 14 Ct difference compared to the RNA backgrounds, representing an apparent 5126 and 163846 higher miRNA amount, respectively. From plasmids encoding other ����non-relevant���� miRNA, very low signals were detected both for miR-1226-3p and miR-33b-5p. The above data indicate that the false positive signals from the relevant plasmid samples are miRNA sequence specific. Therefore, although mature miRNA molecules are not present, signals can be apparently detected from DNA containing the coding sequence of the corresponding pre-miRNA form. These results were also confirmed by experiments using miR-8773p and miR-877-5p assays. Next, we intended to address the question that which part of the measurement misleads the mature miRNA detection. To Diazoxide answer this question, first we made quantitative real-time PCR for miR-1226-3p from reverse transcribed and non-transcribed samples. We tested gDNA and RNA samples from mir-1226 overexpressing cell line and we also used mir-1226 encoding plasmid samples. Mir-33b overexpressing cell line and mir-33b encoding plasmid samples served as nonrelevant controls. As shown in Figure 5A, there is a slight detection during the real-time PCR reaction from the relevant plasmid DNA without reverse transcription, but the majority of the false positive signal is detected only when the reverse transcription reaction is performed. We obtained similar results with the miR-33b-5p assay.In summary, these data reveal the unexpected fact that DNA may serve as a template during the reverse transcription reaction in a sequence specific manner.