T7-based linear amplification of low concentration mRNA samples using beads and microfluidics
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE13595 | GEO | 2009/01/01
SECONDARY ACCESSION(S): PRJNA110707
REPOSITORIES: GEO
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Project description:Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.
2020-09-04 | PXD020857 | Pride
Project description:Epigenome constitutes an important layer that regulates gene expression and dynamics during development and diseases. Extensive efforts have been made to develop epigenome profiling methods using a low number of cells and with high throughput. Chromatin immunoprecipitation (ChIP) is the most important approach for profiling genome-wide epigenetic changes such as histone modifications. In this report, we demonstrate microfluidic ChIPmentation (mu-CM), a microfluidic technology that enables profiling cell samples that individually do not generate enough ChIP DNA for sequencing library preparation. We used a simple microfluidic device to allow 8 samples to be processed simultaneously. The samples were indexed differently using a tagmentation-based approach (ChIPmentation) and then merged for library preparation. Histone modification profile for each individual sample was obtained by demultiplexing the sequencing reads based on the indexes. Our technology allowed profiling 20 cells and is well suited for cell-type-specific studies using low-abundance tissues.
2020-09-22 | GSE152233 | GEO
Project description:The evaluation of zymolyase treatment using yeast gene expression comparison analysis. The yeast BY4743 strain was used for this study. Yeast cells were treated with Zymolyase (final conc. 300 U/ml), and incubated at 37 degree for ten minutes. Digestion process was not provided for control sample. Total RNA was isolated with commercial kit (FastRNA Pro Red kit, Q-Biogene), and amplified RNA (aRNA) was synthesized with 3'IVT Express kit (Affymetrix). All samples were hybridized with Gene Chip (Yeast Genome 2.0 Array, Affymetrix), then each array chip was scanned by GeneChip Sanner 3000 (Affymetrix).
2013-03-21 | GSE36955 | GEO
Project description:Magnetic beads (or particles) having a size between 1 and 5 µm are largely used in many biochemical assays devoted to both purification and quantification of cells, nucleic acids, or proteins. Unfortunately, the use of these beads within microfluidic devices suffers from natural precipitation because of their size and density. The strategies applied thus far to cells or polymeric particles cannot be extended to magnetic beads, mainly due to their magnetization and their higher densities. We report an effective shaking device capable of preventing the sedimentation of beads that are stored in a custom PCR tube. After the characterization of the operating principle, the device is validated for magnetic beads in droplets, leading to an equal distribution between the droplets, barely affecting their generation.
| S-EPMC10304097 | biostudies-literature
Project description:We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-β and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (μ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.
2016-04-10 | GSE59534 | GEO
Project description:Single cell transcriptomics has emerged as a powerful approach to dissecting phenotypic heterogeneity in complex, unsynchronized cellular populations. However, many important biological questions demand quantitative analysis of large numbers of individual cells. Hence, new tools are urgently needed for efficient, inexpensive, and parallel manipulation of RNA from individual cells. We report a simple microfluidic platform for trapping single cell lysates in sealed, picoliter microwells capable of “printing” RNA on glass or capturing RNA on polymer beads. To demonstrate the utility of our system for single cell transcriptomics, we developed a highly scalable technology for genome-wide, single cell RNA-Seq. The current implementation of our device is pipette-operated, profiles hundreds of individual cells in parallel with library preparation costs of ~$0.10-$0.20/cell, and includes five lanes for simultaneous experiments. We anticipate that this system will ultimately serve as a general platform for large-scale single cell transcriptomics, compatible with both imaging and sequencing readouts.!Series_type = Expression profiling by high throughput sequencing A microfluidic device that pairs sequence-barcoded mRNA capture beads with individual cells was used to barcode cDNA from individual cells which was then pre-amplified by in vitro transcription in a pool and converted into an Illumina RNA-Seq library. Libraries were generated from ~600 individual cells in parallel and extensive analysis was done on 396 cells from the U87 and MCF10a cell lines and from ~500 individual cells with extensive analysis on 247 cells from the U87 and WI-38 cell lines. Sequencing was done on the 3'-end of the transcript molecules. The first read contains cell-identifying barcodes that were present on the capture bead and the second read contains a unique molecular identifier (UMI) barcode, a lane-identifying barcode, and then the sequence of the transcript.
2015-05-21 | E-GEOD-66357 | biostudies-arrayexpress
Project description:Assessing the impact of the loss of sample representation caused by the use of global linear amplification (T7-IVT) during microarray hybridization sample preparation.
2009-03-25 | E-MEXP-2099 | biostudies-arrayexpress
Project description:Background: DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system. Results: We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of Pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of Pan-neural RNA. WT-Pico results in a higher fraction of Present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a Reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification. Conclusion: We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting. Keywords: expression profile
2007-11-01 | GSE9485 | GEO
Project description:Dimethyl labeling was integrated into the microfluidic pipeline to allow for precise relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples.
2023-03-24 | PXD036982 | Pride
Project description:The evaluation of mycotoxicity of type B trichothecenes using a yeast gene expression comparison analysis. The yeast BY4743 derivative PDR5 mutant was used for this study. The yeast cells were treated with trichothecene mycotoxins, and incubated at 25 degree for two hours, respectively. Total RNA was isolated with commercial kit (FastRNA Pro Red kit, Q-Biogene), and amplified RNA (aRNA) was synthesized with 3'IVT Express kit (Affymetrix). All samples were hybridized with Gene Chip (Yeast Genome 2.0 Array, Affymetrix), then each array chip was scanned by GeneChip Sanner 3000 (Affymetrix).
2012-09-12 | GSE36954 | GEO