Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Assessing characteristics of RNA amplification methods for single cell RNA sequencing

ABSTRACT: We conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. Our approach was to dilute bulk total RNA (from a single source) to levels bracketing single-cell levels of total RNA (10 pg and 100 pg) in replicates and amplifying the RNA to levels sufficient for RNA sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE88850 | GEO | 2016/10/31

SECONDARY ACCESSION(S): PRJNA348767

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

Project description:For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. Four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. For each protocol, one RNA amplification was performed from 250 pg, and one from 500 pg of human universal RNA by two operators in two independent laboratories and compared to the amplified aRNA obtained from 2 µg and 100 ng RNA inputs following the standard protocol proposed by Affymetrix. A negative control (amplification without total RNA) and a positive control (if available) were included in each experimental batch. Samples indicating 50, 100, and 1000 pg RNA inputs correspond to 3 additional quantities of total RNA used to synthesise the cDNA target using the nugen protocol for comparison (250, 500 pg + 50, 100, 1000 pg).

2009-06-02 | E-GEOD-15398 | biostudies-arrayexpress

Dilution and amplification control study

Project description:Mouse adult female brain’s cortex (C57BL/6, Charles River Laboratories, Inc.) was isolated and stored immediately at -80°C. Subsequently, the mRNA (15µg) was isolated using TRIzol Reagent and MicroFastTrack 2.0 Kit (Invitrogen). A Sample of 5µg was assessed on Affymetrix Mouse 430.2 array. Aliquots from the leftovers of the same cortical mRNA were diluted to single-cell RNA levels (0.1, 1, and 10 pg) and independently aRNA amplified for a total of 2 and 4 rounds and assessed on Affymetrix Mouse 430.2 arrays.

2014-10-14 | GSE61081 | GEO

Dilution and amplification control study

Project description:Mouse adult female brainM-bM-^@M-^Ys cortex (C57BL/6, Charles River Laboratories, Inc.) was isolated and stored immediately at -80M-BM-0C. Subsequently, the mRNA (15M-BM-5g) was isolated using TRIzol Reagent and MicroFastTrack 2.0 Kit (Invitrogen). A Sample of 5M-BM-5g was assessed on Affymetrix Mouse 430.2 array. Aliquots from the leftovers of the same cortical mRNA were diluted to single-cell RNA levels (0.1, 1, and 10 pg) and independently aRNA amplified for a total of 2 and 4 rounds and assessed on Affymetrix Mouse 430.2 arrays. A Sample of 5M-BM-5g of mRNA was assessed on Affymetrix Mouse 430.2 array. Aliquots from the leftovers of the same cortical mRNA were diluted to single-cell RNA levels (0.1, 1, and 10 pg) and independently amplified, as described above, for a total of 2 and 4 rounds and assessed on Affymetrix Mouse 430.2 arrays.

2014-10-14 | E-GEOD-61081 | biostudies-arrayexpress

Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

Project description:ChIP-seq is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, reliably amplifying 50 pg of ChIP DNA, corresponding to ~30,000 input cells for transcription factor ChIP (CEBPA) and 3,000 cells for histone mark ChIP (H3K27me3). This represents a significant advance compared to existing technologies, which involve complex and time-consuming steps of pre-amplification, making them susceptible to experimental biases. ChIP-seq of histone modifications H3K27me3 (2 biological replicates (I+II) , 2 ng input), H3K4me3 (2 biological replicates (II+III), 2 ng input), transcription factor CEBPA (2 biological replicates (I+II), 300 pg input), 4 diluted CEBPA libraries (pool of ChIP from 3 biol. replicates (I+II+III) 3x 100 pg input, 1x 50 pg). Additonal ChIP-seq using 10,000 cells, 1 biological replicate of each H3K4me3 and CEBPA.

2015-02-01 | E-GEOD-55850 | biostudies-arrayexpress

BIGenomics HPV zipcode array multiplex PCR tests 2

Project description:Comparison of two different multiplex PCR primer pools in amplifying target HPV types from plasmid templates. Template concentrations are either 10 pg or 100 pg. Templates are detected by type-spcific LDR probes hybridized on microarray.

2011-08-20 | E-MEXP-3268 | biostudies-arrayexpress

Comparison of Affymetrix and NuGEN amplification methods for HTA 2.0 microarray

Project description:Comparison of total RNA-seq and Affymetrix GeneChip(R) Human Transcriptome Array 2.0 analysis methods and Affymetrix GeneChip® WT PLUS Reagent and NuGEN Ovation® PICO WTA System V2 amplification methods for the detection of significant differentially expressed genes isolated from whole blood and brain RNA samples Affymetrix and NuGEN amplification methods are compared to determine which is most efficient, cost effective, and accurate in the detection of differentialy expressed transcript clusters on the HTA 2.0 microarray The optimum amplification microarray data is compared to total RNA-seq analysis of the same samples to determine which is the most efficient, cost effective, and accurate method of detecting differentially expressed genes

2020-09-30 | GSE120327 | GEO

Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

2015-02-01 | GSE55850 | GEO

BIGenomics HPV zipcode array multiplex PCR tests

Project description:Comparison of two different multiplex PCR primer pools in amplifying target HPV types from plasmid templates. Template concentrations are either 1 ng or 1 pg. Templates are detected by type-spcific LDR probes hybridized on microarray.

2011-05-01 | E-MEXP-3154 | biostudies-arrayexpress

Comparison of genomic DNA amplification methods

Project description:Hybridization of amplified genomic DNA against unamplified genomic DNA using three different amplification methods to assess the bias introduced by amplification alone. Keywords: genomic DNA Three different amplification methods were assessed and total genomic DNA hybridized against equal amounts of amplified genomic DNA for each method.

2010-05-17 | E-GEOD-8449 | biostudies-arrayexpress

Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

Project description:Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known.Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules.Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

| S-EPMC5122016 | biostudies-literature

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