Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy

Abstract

MicroRNAs (miRNAs) play critical roles in controlling various cellular processes, and the expression levels of individual miRNAs can be considerably altered in pathological conditions such as cancer. Accurate quantification of miRNA at the single-cell level will lead to a better understanding of miRNA function. Here, we present a direct and sensitive method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip enabled mature miRNAs, but not premature miRNAs, to be located individually on an adhesion force map. By scanning several sections of a micrometer-sized DNA spot, we were able to quantify the copy number of miR-134 in a single neuron and demonstrate that the expression was increased upon cell activation. Moreover, we visualized individual miR-134s on fixed neurons after membrane removal and observed 2-4 miR-134s in the area of 1.0 × 1.0 μm(2) of soma. The number increased to 8-14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These findings indicate that miRNAs can be reliably quantified at the single cell level with AFM and that their distribution can be mapped at nanometric lateral resolution without modification or amplification. Furthermore, the analysis of miRNAs, mRNAs, and proteins in the same sample or region by scanning sequentially with different AFM tips would let us accurately understand the post-transcriptional regulation of biological processes.

Nanoscale imaging reveals miRNA-mediated control of functional states of dendritic spines

“…All animal care and use were in accordance with the institutional guidelines of Pohang University of Science and Technology. Neuronal cell cultures and AFM imaging were performed as previously described (32). Statistical analysis was performed using IBM SPSS Version 21.0.…”

“…A hybrid binding domain (HBD) binds specifically to RNA/DNA hybrids that do not normally exist in the cytosol (31). We previously took advantage of HBD to detect and quantify miRNAs in single cells (32). Using the HBD with a complementary probe DNA to hybridize with miR-134s as the target, we were able to map individual miR-134s in dendritic shafts and spines at 10-nm resolution (SI Appendix, Fig.…”

Algal Viruses: The (Atomic) Shape of Things to Come

“…For example, multifrequency AFM that can provide more information by measuring several frequencies of cantilever motion such as simultaneous measurement of topography and the viscosity map of virions [ 92 ], cells [ 93 ] and VLPs [ 94 ]. It is also possible to use the AFM as a physical micromanipulator in force nanoindentation studies to map the mechanical properties of viruses in uncoating and capsid interaction studies [ 95 , 96 ] and there exists a modification of force spectroscopy using hybrid binding domain functionalised AFM cantilevers to investigate and quantify the in situ hybridization of miRNA and other nucleic acids in single cells [ 97 ].…”

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

“…173 Recently, studies have shown that AFM can even directly probe RNA in a fixed cell with biochemically sensitive tips. 174 For more descriptions about AFM imaging of DNA, readers are referred to the reviews.…”