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

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

Compact atom source using fiber-based pulsed laser ablation

We designed, demonstrated, and characterized an atom source based on fiber-based pulsed laser ablation. By using commercially available miniature lens system for focusing nanosecond pulsed laser of up to 225~$\mu$J delivered through a multimode fiber of 105~$\mu$m core, we successfully ablate a SrTiO$_3$ target and generate a jet of neutral strontium atoms, though our method can be applied to other transparent ablation targets containing materials under concern. Our device endures 6\,000 cycles of pulse delivery and irradiation without noticeable damage on the fiber facets and lenses. The generated strontium beam is characterized with spectroscopic method and is revealed to exhibit the transverse temperature of 800~K and longitudinal velocity of 2\,300~m/s, which are typical of pulsed-laser-ablation-based atom source. The number of atoms generated by a single ablation pulse is estimated to be $2\times 10^5$. Our device provides a compact, cryo-compatible fiber-pigtailed atom source with minimized device footprints and reduced complexity of vacuum systems to further promote the developments of cold-atom experiments. It may also find interesting applications in atomic and molecular sciences.Comment: 4 pages, 3 figure

All-microwave manipulation of superconducting qubits with a fixed-frequency transmon coupler

All-microwave control of fixed-frequency superconducting quantum computing circuits is advantageous for minimizing the noise channels and wiring costs. Here we introduce a swap interaction between two data transmons assisted by the third-order nonlinearity of a coupler transmon under a microwave drive. We model the interaction analytically and numerically and use it to implement an all-microwave controlled-Z gate. The gate based on the coupler-assisted swap transition maintains high drive efficiency and small residual interaction over a wide range of detuning between the data transmons.Comment: 12 pages, 8 figure

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

Radiation Hybrid Maps of Medaka Chromosomes LG 12, 17, and 22

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was ∼33 kb/cR. We estimate the potential resolution of the RH panel to be ∼186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka

Fast parametric two-qubit gates with suppressed residual interaction using the second-order nonlinearity of a cubic transmon

We demonstrate fast two-qubit gates using a parity-violated superconducting qubit consisting of a capacitively shunted asymmetric Josephson-junction loop under a finite magnetic flux bias. The second-order nonlinearity manifesting in the qubit enables the interaction with a neighboring single-junction transmon qubit via first-order interqubit sideband transitions with Rabi frequencies up to 30 MHz. Simultaneously, the unwanted static longitudinal (ZZ) interaction is eliminated with ac Stark shifts induced by a continuous microwave drive near resonant to the sideband transitions. The average fidelities of the two-qubit gates are evaluated with randomized benchmarking as 0.971, 0.958, and 0.962 for CZ, iswap, and swap gates, respectively

熟練者と非熟練者の表計算ソフトウェアの操作手順

本研究は,熟練者と非熟練者との表計算ソフトウェアの操作手順の違いを明らかにすることを目的とした。熟練者は情報処理の授業を担当する教員3名,非熟練者は情報処理の受講者17名であった。比較のためにDEMATEL法が用いられた。その結果,非熟練者は画面上の見栄えを気にし,表の装飾等の作業を頻繁に繰り返すのに対し,熟練者は文字入力,関数を用いた計算等を順にこなし,最終的に表の整形を行うという傾向が見られた。効率的な作業の進め方についての内容を授業の中に盛り込む必要があると考えられる。This research aimed to find the difference in operating procedure of expert spreadsheet users compared to unskilled users. The subjects of the study were 3 expert teachers of data procesing classes and 17 unskilled users. The DEMATEL method was used for comparison. It was found the unskilled users were overly concerned about the appearance of the spreadsheet; frequently re-inputting data and re-arranging the table. In contrast, expert users input the data, then use functions to do the calculation before finaly working on the appearance of the spreadsheet. It is important that these findings about efficient use of spreadsheets are incorporated into class teaching