scholarly journals Single-cell study of the extracellular matrix effect on cell growth by in situ imaging of gene expression

2017 ◽  
Vol 8 (12) ◽  
pp. 8019-8024 ◽  
Author(s):  
Yupeng Sun ◽  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Xiaojun Ren ◽  
Ling Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Cell Growth ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Matrix Stiffness ◽  
Rolling Circle ◽  
Single Cell Imaging ◽  
Extracellular Matrix Stiffness

The effect of extracellular matrix stiffness on cell growth and the underlying molecular mechanism was investigated using an in situ single-cell imaging of gene expression method based on rolling circle amplification.

scholarly journals SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

2020 ◽  
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

AbstractChanges in cell identities and positions underlie tissue development and disease progression. Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell-states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single Cell Resolution INSitu Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

scholarly journals SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000675
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

scholarly journals Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification

2006 ◽  
Vol 72 (9) ◽  
pp. 6248-6256 ◽  
Author(s):  
Fumito Maruyama ◽  
Katsuji Tani ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Masao Nasu
Keyword(s):  
Single Cell ◽  
Gene Amplification ◽  
Rolling Circle Amplification ◽  
Extracellular Dna ◽  
Single Cell Level ◽  
Dna Uptake ◽  
Cell Level ◽  
Indigenous Bacteria ◽  
Rolling Circle

ABSTRACT Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.

scholarly journals Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Tomasz Krzywkowski ◽  
Sibel Ciftci ◽  
Farzaneh Assadian ◽  
Mats Nilsson ◽  
Tanel Punga
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Single Cell ◽  
Cell Population ◽  
Rolling Circle Amplification ◽  
Expression Patterns ◽  
Human Adenovirus ◽  
Viral Dna ◽  
Rolling Circle ◽  
Infected Cells

ABSTRACT An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

scholarly journals Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization

2021 ◽  
Vol 9 (6) ◽  
pp. 1208
Author(s):  
Kyohei Horio ◽  
Hirokazu Takahashi ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
Tsunehiro Aki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactic Acid ◽  
In Situ Hybridization ◽  
Lactic Acid Bacteria ◽  
Fluorescence In Situ Hybridization ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Microbial Interactions ◽  
Rolling Circle

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton–bacteria interactions.

scholarly journals Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1635
Author(s):  
Ya Su ◽  
Rongxin Fu ◽  
Wenli Du ◽  
Han Yang ◽  
Li Ma ◽  
...  
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Hela Cells ◽  
Quantitative Measurement ◽  
Single Cells ◽  
Dry Mass ◽  
Quantitative Detection ◽  
Label Free ◽  
Mass Sensitivity

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

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