In further experiments, we tested whether the above phenomenon has a real relevance, for example when investigating miRNAs in transiently transfected cells. In such cases, RNA Amphotericin B samples are prepared from cells containing the transfected plasmids. For these measurements we used RNA samples from HeLa cells transiently transfected with different amounts of a mir1226 encoding plasmid. The data showed that when samples were not treated with DNase, a significantly higher amount of miRNA was detected as compared to the DNase treated samples. This problem occurred not only by using the Trizol based total RNA isolation method, but also when applying a column-based isolation protocol such as the mirVana Kit. These results indicate that there is plasmid DNA contamination in the total RNA samples which indeed misleads the Fenofibric acid accurate detection of mature miRNAs. In this study, we examined several factors in detail influencing accuracy and reliability of the miRNA quantitative stem-loop PCR. Considering the reverse transcription step of this methodology, our data indicate that the increase of the total RNA amount can result in a lower apparent miRNA expression level. This phenomenon could occur due to dissimilar reaction efficiencies of the target and the control in certain ranges of total RNA amount. Thus, it may lead to elevated detection of the endogenous control compared to the target, therefore resulting in an apparent decrease in the level of the target. For a particular endogenous control/target pair the optimal amount of the total RNA for the reverse transcription reaction can vary, therefore pilot investigations are advisable prior to the real experiments. However, based on our experiments, 10�C20 ng of total RNA might be adequate. In addition to these data, we provided evidence that the target of interest can be reverse transcribed together in one reaction with the appropriate endogenous control. Apart from lowering the costs of experiments, it has the advantage of reducing pipetting errors and thereby making the measurements more accurate. Next, we found the unexpected result that contrary to the claims of the original protocol, DNA could serve as a template during mature miRNA measurements, mostly during the reverse transcription reaction.