Ontology highlight
ABSTRACT:
PROVIDER: PRJNA94845 | ENA |
REPOSITORIES: ENA
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Project description:A systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. Keywords: Method validation
2006-04-26 | GSE4308 | GEO
Project description:A systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. Experiment Overall Design: To verify the ability of the developed method, we prepared total RNA from ES cells and diluted it to the single-cell level (10 pg). Eight array data from the cDNA samples amplified independently from the 10 pg RNA (amplified samples) were compared to each other, and to eight array data from the undiluted 5 ug RNA (nonamplified controls). Since all the materials were the same, all of the observed variations were attributable to the methodogoly.
2008-06-13 | E-GEOD-4308 | biostudies-arrayexpress
Project description:Expression data for validation of single cell cDNA amplification method (V1V3 method)
| PRJNA104647 | ENA
Project description:We have developed a method of mRNA amplification based on T7 polymerase, where T7 promoter is attached to a oligonucleotide containing the mini-exon sequence, which will hybridize with cDNA molecules containing the anti-ME sequence
2017-07-31 | GSE94766 | GEO
Project description:Background: With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes the development of this method and its application to mapping transcriptome profiles. Results: RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. In addition, five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were artifacts. Conclusions: An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is more sensitive in detecting small amount of RNA compared to end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.
2010-12-09 | GSE25876 | GEO
Project description:Spatially resolved, single-cell transcriptome analysis of high quality remains challenging, despite the rise of high-resolution spatial transcriptomics. Laser-capture microdissection (LCM) is widely used to isolate cells of interest from tissue sections. Here, we developed DRaqL (Direct RNA recovery and quenching for LCM), a technique that allows efficient lysis of single, LCM-isolated cells from alcohol- and formalin-fixed tissue and stained frozen sections, and that is amenable to enzymatic reactions for cDNA amplification within the same sampling tubes. Quantitative evaluations showed that single-cell RNA sequencing combined with DRaqL allowed transcriptomic profiling from frozen sections at an efficiency comparable with that from freshly dissociated cells, with small biases and errors in lowly expressed genes, in addition to allowing exon-exon junction profiling. By applying this method to mouse ovarian frozen sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with the size of oocytes and follicles, identified genes highly correlated with oocyte diameters, and detected oocyte-specific splice isoforms. We further identified genes that were differentially expressed in granulosa cells depending on their distance from oocytes, suggesting distinct epigenetic regulatory mechanisms and cellular proliferation. Thus, DRaqL provides an efficient cell lysis for single-cell cDNA amplification from frozen sections that is amenable to high-quality RNA sequencing analysis.
2023-09-01 | GSE192551 | GEO
Project description:Improved detection of global copy number variation using high density, non-polymorphic oligonucleotide probes
| PRJNA102563 | ENA
Project description:We developed a method, Reverse Transcription with Random Displacement Amplification (RT-RamDA), to reverse transcribe and amplify cDNA directly from RNA templates using strand displacement and RNA-dependent DNA polymerase activity. This method resulted in a cDNA yield approximately 100-fold higher than conventional RT with high sensitivity, enabling gene expression profiling of low-expression genes with a small amount of RNA, such as that obtained at the single-cell level.
2019-01-01 | GSE108463 | GEO
Project description:Single cell sequencing (SCS) is a promising approach for precisely digging into the genetic heterogeneity at single cells level. Single cell whole genome/exome sequencing (scWGS/scWES) and single cell whole transcriptome sequencing (scRNA-seq) are methods of SCS and have been applied in various studies of solid tumors, e.g. liver cancer, breast cancer. However, no scRNA-seq-related studies of human colon-cancer tissue-samples have been conducted. In this study, we developed a modified and efficient colon-cancer-tissue SCSs system (named CCTSs-SCSs system) by combining four optimization technologies: tissue digestion, live and intact single-cells capture, single-cell RNA or genome amplification and sequencing, and bioinformatics analysis. This advanced system is applicable to both scRNA-seq and scWGS/scWES. Using this system, we successfully completed several single cells scRNA-seq and scWES, and found three key points of this system: an improved digestion system, FACS-based dead-cell-removal method for scRNA-seq, 20 cycles for scRNA amplification. Our CCTSs-SCSs system provides a reliable and efficient method of scRNA-seq or scWGS/scWES for human colon cancer tissue and contributes to single-cell level and precise studies for colon cancer.
2019-12-01 | GSE92432 | GEO
Project description:Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility, and cost-efficiency can advance many research questions. Among the flexible plate-based methods, single-cell RNA barcoding and sequencing (SCRB-seq) is highly sensitive and efficient. Here, we systematically evaluate experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a scRNA-seq library protocol we call molecular crowding SCRB-seq (mcSCRB-seq), which we show to be one of the most sensitive, efficient, and flexible scRNA-seq methods to date.
2018-07-26 | GSE103568 | GEO