We have performed the siRNA-mediated knockdown of Dicer protein partners and not their knockout, therefore some portion of these proteins remained in the cells. These proteins could form a smaller amount of the fully functional Dicer complex. Therefore, only a portion of pre-miRNAs could be processed and the rest underwent degradation. As a result, we observed smaller amounts of pre-miRNAs and miRNAs. Our experiments revealed that the silencing of Dicer protein partners had mostly a minor effect on the specificity of Dicer cleavages. The distribution of the length variants of miRNAs differed slightly between the analyzed samples and the control. The changes in the Dicer cleavage pattern could result from the activity of the RNase fraction having changed cleavage specificity when deprived of either protein partner. The transfection of synthetic pre-miRNAs combined with the northern blotting analysis may be considered another experimental approach to study the Dicer step of miRNA biogenesis. This system, however, needs improvement to make it applicable to a larger number of pre-miRNAs. The synthetic miRNA precursors, used in these experiments, differ in their sequence and structure and may not be equally good substrates for binding and cleavage by the endogenous Dicer complex. It is unlikely that such precursors are fully captured by Dicer. Their substantial portion may be trapped in endosomes. The fraction that escapes endosomes may enter the miRNA biogenesis pathway or may become a target for cellular exo- and endoribonucleases. We do not know what the contribution is of such unspecific processes to the miRNA patterns observed in Figure 4A. We propose, however, that this contribution is not very large because most of the observed northern blot signals belonged to undegraded precursors and miRNA sized products. Recent research shows that siRNAs transfected to cells are primarily intercepted by Dicer, which recognizes 2nt 39-overhangs, but the efficiency of capture of different siRNAs is highly variable and dependent on the nature of the 39 overhang sequence. It is also likely that synthetic miRNA precursors transfected to cells may be primarily captured by Dicer that is present in the RLC complex, but the LDK378 affinity of the Dicer complex to different pre-miRNAs may vary greatly. The single-nick intermediate is observed in various amounts for various substrates when synthetic precursors are cleaved with recombinant Dicer. In contrast, in HeLa cells transfected with synthetic pre-miRNAs or miRNA-encoding plasmids, the intermediate product was not observed, suggesting the potential role of protein partners in the synchronization of cleavages generated by individual RNase III domains of Dicer. We considered two alternatives to explain the easy detection of the single-nick intermediates in reactions with recombinant Dicer and the inability to detect this cleavage intermediate in cellular systems. One possibility is that the endogenous Dicer and its protein partners may release the single-nicked precursor to the cytosol, where it undergoes rapid degradation by ribonucleases and escapes detection. The possibility which we consider more likely is that the cleavages generated by two RNase III domains of Dicer are much better synchronized.