mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

<p>Abstract</p> <p>Background</p> <p>The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. <it>In situ </it>hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.</p> <p>Results</p> <p>In order to evaluate the SCN method, we compared the SCN method with <it>in situ </it>hybridization (ISH). First, we examined spatial β-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for β-actin mRNA. In the SCN method, quantity of β-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of β-actin mRNA. We showed that intensity of ISH is higher; quantity of β-actin mRNA detected by the SCN method increased more.</p> <p>Conclusion</p> <p>In this study, we compare the SCN method with the ISH. We examined β-actin mRNA expression in single cells using both methods. We picked up β-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.</p

mRNA analysis of single living cells

Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM). AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells

Interaction between Pheromone and Its Receptor of the Fission Yeast Schizosaccharomyces pombe Examined by a Force Spectroscopy Study

Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 μm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor

Effect of Sintering Parameters on Physical Properties of SS 316L Alloy Compact / Mohd Faizul Farhan Ahmad Hamidi..[et al.]

Recently, metal injection moulding (MIM) has been identified as an economical fabrication technique for metal components. MIM, adapted from plastic injection moulding, has four major processing stages, which are: mixing of powder and binder system to produce feedstock, injection moulding, debinding and sintering. In this paper, the physical properties of SS 316L alloy sintered compact sintered at three different sintering parameters were discussed. Thermal properties of the feedstock were characterized by Thermogravimetric Analyzer (TGA) analysis. The debound compacts were sintered at 1100, 1200, and 1300 °C for 3 hours in the high pure argon atmosphere. The heating rate has been set at 10°C/min. The relative density, dimensional shrinkage and microstructure of the sintered compact were investigated. The relative density and dimensional shrinkage increased with the sintering parameters due to high densification of the sintered compact. Some pore sizes seemed to decrease with the increase in sintering temperature. Different sintering temperature shows a significant effect on the physical properties of the sintered compact

A review of the Japanese species of the choragella - group of the genus Morophaga Herrich-Schäffer (Lepidoptera: Tineidae), with description of a new species

Osada, Yohei, Hirowatari, Toshiya (2016): A review of the Japanese species of the choragella - group of the genus Morophaga Herrich-Schäffer (Lepidoptera: Tineidae), with description of a new species. Zootaxa 4127 (1): 192-200, DOI: http://doi.org/10.11646/zootaxa.4127.1.1

The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, the mam2 upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter

A review of the Japanese species of the choragella - group of the genus Morophaga Herrich-Schäffer (Lepidoptera: Tineidae), with description of a new species

Osada, Yohei, Hirowatari, Toshiya (2016): A review of the Japanese species of the choragella - group of the genus Morophaga Herrich-Schäffer (Lepidoptera: Tineidae), with description of a new species. Zootaxa 4127 (1): 192-200, DOI: http://doi.org/10.11646/zootaxa.4127.1.1