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ۼ : 16-08-17 17:48
[2016.08]ؿ Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy
 ۾ :
ȸ : 855  


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




Hyunseo Koo, Ikbum Park, Yoonhee Lee, Hyun Jin Kim, Jung Hoon Jung, Joo Han Lee, Youngkyu Kim, Joung-Hun Kim and Joon Won Park

 


 
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 micron-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 mem-brane removal and observed 2–4 miR-134s in the area of 1.0
1.0 m2 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

 




 
̰ ȣۿ ü ϰ, پ ִ microRNA ΰ м ִ Ͽ. а Ƿκ RNA/DNA duplex ִ hybrid binding domain (HBD) ޾, ڰ Ǿ ִ AFM cantilever Ͽ force-mapping miRNA м Ȯ Ͽ, ڱ , miR-134 þ ̹ Ȯ Ͽ.

  ȭ Ǿ ִ ȿ (reverse transcription-polymerase chain reaction, RT-PCR) ̿ biomarker м ΰ ̰ , miRNA strand ª ̷ Ͽ ÿ ִµ, ع̷ ϴ miRNA (~ 500 ea) , Ȯ ִٴ ִ. Ȱȴٸ, Ŀ Ը о߿ ϰ Ȱ ȴ. 2016 8, ̱ ȭȸ (Journal of the American Chemical Society) Ǿ.


 
 

Total 77
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