Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling

ABSTRACT: To correlate gene expression profiles to fundamental biological processes such as cell growth, differentiation and migration, it is essential to work at the single cell level. Gene expression analysis always starts with the relatively low efficient reverse transcription (RT) of RNA into complementary DNA (cDNA), an essential step as unprocessed RNAs will not be analyzed further. In this paper, we present a novel method for RT that uses microfluidics to manipulate nanoliter volumes. We compare our method to conventional protocols performed in microliter volumes. More specifically, reverse transcription was performed either in a poly-dimethylsiloxane (PDMS) rotary mixer or in a tube, using single cell amount of mouse brain RNA (10 pg), and was followed by a template-switching PCR (TS-PCR) amplification step. We demonstrate that, using the microfluidic protocol, 74% of the genes expressed in mouse brain were detected, while only 4% were found with the conventional approach. We next profiled single neuronal progenitors. Using our microfluidic approach, i.e. performing cell capture, lysis and reverse transcription on-chip followed by TS-PCR amplification in tube, a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method. Keywords: Single cell, transcriptome, PDMS, microfluidic, reverse-transcription (RT), template-switching, lab-on-a-chip

ORGANISM(S): Mus musculus

PROVIDER: GSE6941 | GEO | 2008/02/15

SECONDARY ACCESSION(S): PRJNA98123

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Comparison of RNA Amplification Techniques meeting the demands for the Expression Profiling of Clinical Cancer Samples

Project description:For microarray experiments starting with nanogram amounts of RNA it is essential to implement reproducible and powerful RNA amplification techniques. Available methods were mainly tested for reproducibility, only a few studies concentrated on potential amplification bias. We evaluated three amplification protocols, which are less time-consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template Switching (TS)-PCR (SMART-PCR Kit, BD), Ribo-SPIA (single primer isothermal amplification, Oviation, Nugen) and a random primer-based PCR. Additionally a more sensitive labeling method, Dendrimer-labeling (Genisphere), was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. Hybridizations were carried out on a targeted two-colour oligonucleotide microarray. From our results we conclude that RNA amplification with TS-PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with TS-PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. According to our experiments TS-PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number (RIN) of 4.3. Keywords: microarray expression profiling, RNA amplification techniques, RNA integrity

2007-08-24 | GSE5588 | GEO

Quantification of RNA editing in the Drosophila Genetic Reference Panel

Project description:We report the application of mmPCR-seq to male whole body samples from 131 strains of the DGRP. We quantified RNA editing at 605 different loci using a microfluidic multiplex PCR method coupled with deep sequencing. RT-PCR amplification of 605 loci from 131 fly strains to quantify RNA editing levels.

2015-08-06 | E-GEOD-67082 | biostudies-arrayexpress

A novel global cDNA amplification technology directly from an RNA template via reverse transcription

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

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

Project description:We report a novel single-cell total RNA-seq method, RamDA-seq, by combining a novel reverse transcription (RT) technology, RT with random displacement amplification (RT-RamDA), and not-so-random (NSR) primers. RT-RamDA provides global cDNA amplification directly from RNA during RT without any universal adopters, which benefits RT efficiency, simplification of the procedure, avoiding the step of PCR amplification, and decontamination of genomic DNA. NSR enables random priming while preventing cDNA synthesis from rRNAs. RamDA-seq showed high sensitivity to non-poly(A) RNAs and full-length coverage even for extremely long transcripts (>10 kb). Moreover, RamDA-seq revealed recursive splicing, a multi-step, splicing, within > 300 kbp-long introns. Finally, RamDA-seq enabled the first genome-wide analysis of enhancer RNAs (eRNAs) in single cells.

2018-02-12 | GSE98664 | GEO

The transcriptome profiling of multi-cell types by the droplet based full-length single-cell transcript sequencing

Project description:We developed a scalable Tn5-based Full-Length In situ Transcriptome sequencing method (scFLIT-seq). This method combines the advantages of in situ reverse transcription of methanol-fixed cells, single-cell separation and barcode ligation via the 10X scATAC-seq protocol and microfluidic platform, and Tn5-mediated fragmentation and tagging of cDNA. Various cells treated with different concentrations of methanol were used to validate the efficacy of this method. The efficacy at the single-cell level of this method was validated on a mixed cell population of HEK293T and NIH/3T3 cells.

2024-01-01 | GSE233170 | GEO

A diffusion-based microfluidic device for single-cell RNA-seq

Project description:Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-Seq). Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one microfluidic chamber. MID-RNA-Seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of MID-RNA-Seq device will be important for transcriptomic studies of scarce cell samples.

2019-02-25 | GSE119271 | GEO

Real-time quantitative PCR analysis of mouse lungs after exposure to early SARS-CoV-2 bearing 614G, Kappa or Delta variant

Project description:Age-matched K18-hACE2 transgenic mice were passively transferred with human COVID-19 mRNA post-vaccination (post-vac) sera followed by the lethal challenge with different SARS-CoV-2 viruses via intranasal route, including (1) New York-PV09158/2020 (NY) of lineage B.1.3 bearing 614G, (2) Kappa (B.1.617.1) and (3) Delta (B.1.617.2). Mouse lungs were harvested on 5 days post infection (dpi). Total RNA was extracted using QIAgen RNeasy Plus Mini Kit and was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Resultant cDNA was used as the template along with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) to perform RT² Profiler™ PCR Array Mouse Hypoxia Signaling Pathway (Qiagen) real-time PCR in Stratagene MX3000p qPCR system.

2022-11-21 | GSE193839 | GEO

Real-time quantitative PCR analysis of mouse lungs after exposure to SARS-CoV-2 and variants of concern (VOC)

Project description:Age-matched K18-hACE2 transgenic mice were infected intranasally with different SARS-CoV-2 viruses, including (1) USA-WA1/2020 (WA) of lineage A, (2) New York-PV09158/2020 (NY) of lineage B.1.3, (3) USA/CA_CDC_5574/2020 (CA) of lineage B.1.1.7 and (4) hCoV-19/South Africa/KRISP-EC-K005321/2020 (SA) of lineage B.1.351. Mouse lungs were harvested on 3 days post infection (dpi). Total RNA was extracted using QIAgen RNeasy Plus Mini Kit and was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Resultant cDNA was used as the template along with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) to perform RT² Profiler™ PCR Array Mouse Hypoxia Signaling Pathway (Qiagen) real-time PCR in Stratagene MX3000p qPCR system.

2021-10-10 | GSE183621 | GEO

A Highly Sensitive and Specific System for Large-scale Gene Expression Profiling

Project description:Gene expression profiling of very few or even single cells is of particular interest in many applications. However, detection of a large number of mRNA sequences from a small number of cells is limited by the sensitivity of available methods. High-throughput multiplex reverse transcription followed by PCR amplification (RT-PCR) has much to offer to these studies due to its inherent sensitivity, efficiency and cost-effectiveness. A multiplex RT-PCR based high-throughput gene profiling system is described in this communication. With this system >1000 different mRNA species can be amplified in a single tube to a detectable amount. By using specially designed PCR primers, the long-standing low specificity problem associated with high-throughput gene expression profiling has been solved. Amplified sequences are then resolved by microarray with probes that only hybridize to sequences amplified from mRNA. The method is so sensitive that mRNA in single cells can be reliably detected. Differentially expressed genes identified with the high-throughput approach in the breast cancer cell line, MCF-7, and its drug resistant variant, MCF-7/AdrR, could be validated by a different method. The approach may greatly facilitate the analysis of combinatorial expression of known genes in any cells in many important applications with a limited amount of RNA. Keywords: drug resistence

2007-06-01 | GSE5920 | GEO

GSC RT-PCR amplification of 10 cells (SP & CD8 T cells), single SP cell and single-SP-cell equivalent

Project description:GSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48333-GSM48344: Single SP cell from C57Bl/6 male mouse bone marrow were sorted into individual wells of 96-well plates. The mRNA of these single cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48345-GSM48349: Forty bone marrow SP cells from C57Bl/6 male mouse bone marrow, Sca-1 positive and Gr-1 negative, gated on the tip of the SP tail, were sorted into 160 microliters of lysis buffer (40 times the amount used for single cells). 4-microliter aliquots (containing the mRNA equivalent to one single cell) were dispensed into individual wells of 96-well plates. The mRNA contained in each aliquot was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. Detection of the microarray hybridization signals was done according to the standard Affymetrix protocol (antibody amplified). Keywords = HSC Keywords = stem cell Keywords = SP Keywords = side population Keywords = CD8 Keywords = lymphocytes Keywords = global single cell RT-PCR Keywords = GSC RT-PCR Keywords: parallel sample

2006-01-02 | GSE2534 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data