scholarly journals Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

2008 ◽  
Vol 36 (15) ◽  
pp. e92-e92 ◽  
Author(s):  
Takahiro Tougan ◽  
Daisuke Okuzaki ◽  
Hiroshi Nojima
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Pcr Amplification ◽  
High Quality

scholarly journals High-Quality Draft Single-Cell Genome Sequence Belonging to the Archaeal Candidate Division SA1, Isolated from Nereus Deep in the Red Sea

2018 ◽  
Vol 6 (19) ◽  
pp. e00383-18 ◽  
Author(s):  
David K. Ngugi ◽  
Ulrich Stingl
Keyword(s):  
Single Cell ◽  
Lake Sediments ◽  
Red Sea ◽  
Genome Sequence ◽  
Soda Lake ◽  
Genome Sequences ◽  
High Quality ◽  
Candidate Division ◽  
Cell Genome ◽  
Single Cell Genome

ABSTRACT Candidate division SA1 encompasses a phylogenetically coherent archaeal group ubiquitous in deep hypersaline anoxic brines around the globe. Recently, the genome sequences of two cultivated representatives from hypersaline soda lake sediments were published. Here, we present a single-cell genome sequence from Nereus Deep in the Red Sea that represents a putatively novel family within SA1.

scholarly journals Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

2019 ◽  
Author(s):  
Suraj Kannan ◽  
Matthew Miyamoto ◽  
Brian Lin ◽  
Renjun Zhu ◽  
Sean Murphy ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Sorting ◽  
Large Particle ◽  
Fluorescence Activated Cell Sorting ◽  
High Quality ◽  
Adult Cardiomyocytes ◽  
Single Cell Rna Sequencing ◽  
Mitochondrial Transcripts ◽  
Manual Methods

ABSTRACTRationaleSingle cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs).ObjectiveWe investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs.Methods and ResultsWe found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties.ConclusionsOur study enables high quality dissection of adult CM biology at single-cell resolution.

scholarly journals Single-cell RNA sequencing of human kidney

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Jinling Liao ◽  
Zhenyuan Yu ◽  
Yang Chen ◽  
Mengying Bao ◽  
Chunlin Zou ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Biology ◽  
Kidney Diseases ◽  
Human Kidney ◽  
High Quality ◽  
Renal Tubular Cells ◽  
Single Cell Rna Sequencing ◽  
Normal Human ◽  
Renal Tubular

AbstractA comprehensive cellular anatomy of normal human kidney is crucial to address the cellular origins of renal disease and renal cancer. Some kidney diseases may be cell type-specific, especially renal tubular cells. To investigate the classification and transcriptomic information of the human kidney, we rapidly obtained a single-cell suspension of the kidney and conducted single-cell RNA sequencing (scRNA-seq). Here, we present the scRNA-seq data of 23,366 high-quality cells from the kidneys of three human donors. In this dataset, we show 10 clusters of normal human renal cells. Due to the high quality of single-cell transcriptomic information, proximal tubule (PT) cells were classified into three subtypes and collecting ducts cells into two subtypes. Collectively, our data provide a reliable reference for studies on renal cell biology and kidney disease.

scholarly journals Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Masato Kogawa ◽  
Masahito Hosokawa ◽  
Yohei Nishikawa ◽  
Kazuki Mori ◽  
Haruko Takeyama
Keyword(s):  
Single Cell ◽  
High Quality

scholarly journals Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell

2003 ◽  
Vol 49 (3) ◽  
pp. 415-424 ◽  
Author(s):  
James M Gale ◽  
Christopher P Romero ◽  
Gregory B Tafoya ◽  
Jérôme Conia
Keyword(s):  
Dna Sequencing ◽  
Single Cell ◽  
Optical Trapping ◽  
Pcr Amplification ◽  
P53 Gene ◽  
Rrna Gene ◽  
Dna And Rna ◽  
A Cell ◽  
Nonadherent Cells ◽  
Positive Results

Abstract Background: Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR. Methods: Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing. Results: Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)n length polymorphism in p53 introns. Conclusions: These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.

High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass

2016 ◽  
Vol 187 ◽  
pp. 341-352 ◽  
Author(s):  
David Perez-Guaita ◽  
Dean Andrew ◽  
Philip Heraud ◽  
James Beeson ◽  
David Anderson ◽  
...  
Keyword(s):  
Single Cell ◽  
Numerical Aperture ◽  
Single Cell Level ◽  
Cell Level ◽  
High Quality ◽  
Pixel Resolution ◽  
Ftir Microscopy ◽  
Synchrotron Source ◽  
Average Spectrum ◽  
Highly Sensitive

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

scholarly journals Differential Gene Expression in Individual Papilla-Resistant and Powdery Mildew-Infected Barley Epidermal Cells

2004 ◽  
Vol 17 (7) ◽  
pp. 729-738 ◽  
Author(s):  
Torben Gjetting ◽  
Timothy L. W. Carver ◽  
Leif Skøt ◽  
Michael F. Lyngkjær
Keyword(s):  
Gene Expression ◽  
Powdery Mildew ◽  
Single Cell ◽  
Pcr Amplification ◽  
Epidermal Cells ◽  
Powdery Mildew Fungus ◽  
Pathogenesis Related Protein ◽  
Infected Cells ◽  
Transcript Profiles ◽  
Microarray Expression

Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. hordei) is determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells defend themselves against attempted fungal penetration by localized responses leading to papilla deposition and reinforcement of their cell wall. This conveys a race-nonspecific form of resistance. However, this defense is not complete, and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole leaves or even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from individual barley epidermal cells was established and proven useful for analyzing resistant and successfully infected cells separately. Contents of single epidermal cells (resistant, infected, and unattacked controls) were collected, and after cDNA synthesis and PCR amplification, the resulting sample was hybridized to dot-blots spotted with genes, including some previously reported to be induced upon pathogen attack. Transcripts of several genes, (e.g., PR1a, encoding a pathogenesis related protein, and GLP4, encoding a germin-like protein) accumulated specifically in resistant cells, while GRP94, encoding a molecular chaperone, accumulated in infected cells. Thus, the single-cell method allows discrimination of transcript profiles from resistant and infected cells. The method will be useful for microarray expression profiling for simultaneous analysis of many genes.

The effects of preservatives and temperatures on arachnid DNA

2005 ◽  
Vol 19 (2) ◽  
pp. 99 ◽  
Author(s):  
Cor J. Vink ◽  
Steven M. Thomas ◽  
Pierre Paquin ◽  
Cheryl Y. Hayashi ◽  
Marshal Hedin
Keyword(s):  
Propylene Glycol ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
The Other ◽  
Dna Fragments ◽  
Cytochrome Oxidase Subunit ◽  
High Quality ◽  
C Storage ◽  
Time Period ◽  
Better Than

We tested the effects of different preservatives and temperatures on the yield of spider and scorpion DNA useable for PCR amplification. Our experiment was designed to simulate conditions in the field and laboratory over a six-week time period, testing the preservatives RNAlater®, propylene glycol, and various ethanol concentrations. Three replicates of each preservation treatment were stored at five different temperature treatments; –80°C, –20°C, 2–4°C, 19–24°C, and 40°C. DNA was extracted and quality was assessed by electrophoresis on mini-gels, and by PCR amplification of high copy mitochondrial DNA fragments (cytochrome oxidase subunit I) and low copy nuclear DNA fragments (actin). Results show that RNAlater® and propylene glycol are significantly better than the other preservatives for high quality DNA preservation and that tissue is best stored at –80°C or –20°C. Storage in 95% ethanol is appropriate if specimens are stored at –20°C or –80°C. We believe our results can help guide biologists in choosing preservatives and temperatures for DNA-based research on arachnids, other arthropods and invertebrates in general.

Sign in / Sign up

Export Citation Format

Share Document