Transfection of microRNA Mimics Should Be Used with Caution

Abstract

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

MicroRNA-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes

“…7i). Importantly, we performed and validated many of our biological studies using lentiviral-mediated overexpression, which does not cause supraphysiological levels of miRNA or accumulation of artefactual RNA species50. Thus, constitutive lentiviral-mediated miR-141 expression, which led to a slight increase in miR-141 copy numbers, impressively inhibited clonal and clonogenic capacities of primary HPCa cells (Fig.…”

“…Some functional studies herein were conducted by transfecting PCa cells with 30 nM synthetic miR-141 oligos and miRNA mimic transfection has been shown to cause ‘supraphysiological' levels of intracellular miRNA and accumulation of artifactual RNA species50. Indeed, transfection of 30 nM of miR-141 in PCa cells can, in a cell type-dependent manner, result in >100–67,000-fold increase in miR-141 levels compared with NC transfected cells.…”

Toward the promise of microRNAs – Enhancing reproducibility and rigor in microRNA research

“…However, one must be aware of potential problems. For example, abnormal AGO loading and formation of high molecular weight aggregates have been reported in transfection experiments, 52 while the presence of microRNA inhibitors has been found to directly inhibit qPCR reactions. Length-dependent activation of dsRNA sensor pathways by microRNA mimics (but not by commercially available 21-nucleotide controls) can also cloud interpretation of results.…”

Genomic conservation of erythropoietic microRNAs (erythromiRs) in white-blooded Antarctic icefish

“…Injection of each of these three miRNAs into zebrafish embryos reduced the production of red blood cells (Xu et al, 2015), but other developmental defects also occurred that can appear due to non-specific deleterious effects of over-expression experiments (Jin et al, 2015; Zhang et al, 2013). Despite their interesting findings and important dataset, these authors did not examine the global conservation of erythromiRs in icefish, leaving open the possibility that icefish genomes may have lost some key erythromiR genes that are required for normal erythropoiesis or erythroid maturation.…”