Length bias-free extraction of cytoplasmic RNA from single cells by electrical lysis and electrophoresis

Author(s):  
Mahmoud N. Abdelmoez ◽  
Yusuke Oguchi ◽  
Yuka Ozaki ◽  
Ryuji Yokokawa ◽  
Hidetoshi Kotera ◽  
...  
2015 ◽  
Vol 2015 (0) ◽  
pp. _J0540101--_J0540101-
Author(s):  
Shota HATA ◽  
Ryuji YOKOKAWA ◽  
Hidetoshi KOTERA ◽  
Hirofumi SHINTAKU

2021 ◽  
Vol 118 (38) ◽  
pp. e2106353118
Author(s):  
Yue Wu ◽  
Afu Fu ◽  
Gilad Yossifon

Herein, we studied localized electroporation and gene transfection of mammalian cells using a metallodielectric hybrid micromotor that is magnetically and electrically powered. Much like nanochannel-based, local electroporation of single cells, the presented micromotor was expected to increase reversible electroporation yield, relative to standard electroporation, as only a small portion of the cell’s membrane (in contact with the micromotor) is affected. In contrast to methods in which the entire membrane of all cells within the sample are electroporated, the presented micromotor can perform, via magnetic steering, localized, spatially precise electroporation of the target cells that it traps and transports. In order to minimize nonselective electrical lysis of all cells within the chamber, resulting from extended exposure to an electrical field, magnetic propulsion was used to approach the immediate vicinity of the targeted cell, after which short-duration, electric-driven propulsion was activated to enable contact with the cell, followed by electroporation. In addition to local injection of fluorescent dye molecules, we demonstrated that the micromotor can enhance the introduction of plasmids into the suspension cells because of the dielectrophoretic accumulation of the plasmids in between the Janus particle and the attached cell prior to the electroporation step. Here, we chose a different strategy involving the simultaneous operation of many micromotors that are self-propelling, without external steering, and pair with cells in an autonomic manner. The locally electroporated suspension cells that are considered to be very difficult to transfect were shown to express the transfected gene, which is of significant importance for molecular biology research.


2019 ◽  
Vol 10 (5) ◽  
pp. 1506-1513 ◽  
Author(s):  
Min Li ◽  
Robbyn K. Anand

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps.


2019 ◽  
Vol 92 (1) ◽  
pp. 1485-1492
Author(s):  
Mahmoud N. Abdelmoez ◽  
Yusuke Oguchi ◽  
Yuka Ozaki ◽  
Ryuji Yokokawa ◽  
Hidetoshi Kotera ◽  
...  
Keyword(s):  

2015 ◽  
Vol 36 (14) ◽  
pp. 1658-1662 ◽  
Author(s):  
Kentaro Kuriyama ◽  
Hirofumi Shintaku ◽  
Juan G. Santiago
Keyword(s):  

2018 ◽  
Vol 90 (21) ◽  
pp. 12512-12518 ◽  
Author(s):  
Sangamithirai Subramanian Parimalam ◽  
Yusuke Oguchi ◽  
Mahmoud N. Abdelmoez ◽  
Arata Tsuchida ◽  
Yuka Ozaki ◽  
...  

2021 ◽  
Vol 7 (15) ◽  
pp. eabe0317
Author(s):  
Yusuke Oguchi ◽  
Yuka Ozaki ◽  
Mahmoud N. Abdelmoez ◽  
Hirofumi Shintaku

Alternative mRNA isoforms play a key role in generating diverse protein isoforms. To dissect isoform usage in the subcellular compartments of single cells, we introduced an novel approach, nanopore sequencing coupled with single-cell integrated nuclear and cytoplasmic RNA sequencing, that couples microfluidic fractionation, which separates cytoplasmic RNA from nuclear RNA, with full-length complementary DNA (cDNA) sequencing using a nanopore sequencer. Leveraging full-length cDNA reads, we found that the nuclear transcripts are notably more diverse than cytoplasmic transcripts. Our findings also indicated that transcriptional noise emanating from the nucleus is regulated across the nuclear membrane and then either attenuated or amplified in the cytoplasm depending on the function involved. Overall, our results provide the landscape that shows how the transcriptional noise arising from the nucleus propagates to the cytoplasm.


2019 ◽  
Vol 59 (2) ◽  
pp. 088-090
Author(s):  
Yusuke OGUCHI ◽  
Mahmoud Nady ABDELMOEZ ◽  
Hirofumi SHINTAKU
Keyword(s):  

Author(s):  
Glenn M. Cohen ◽  
Radharaman Ray

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.


Author(s):  
J. H. Luft

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).


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