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Non-biased and efficient global amplification of a single-cell cDNA library.

ABSTRACT: Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.

SUBMITTER: Huang H

PROVIDER: S-EPMC3902946 | biostudies-literature |

REPOSITORIES: biostudies-literature

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An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

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| S-EPMC1409679 | biostudies-literature

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

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An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

Project description:This SuperSeries is composed of the SubSeries listed below.

2006-03-20 | GSE4309 | GEO

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A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol.

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An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

Project description:A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness.

| S-EPMC4725349 | biostudies-literature

Expression data for validation of single cell cDNA amplification method (V1V3 method)

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

Characterization of an Atlantic cod (Gadus morhua) embryonic stem cell cDNA library.

Project description:BackgroundThe Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES) cells, our goal was to generate and analyze expressed sequence tags (ESTs) from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs.ResultsWe sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences). BLASTX searches produced 889 significant (E-value < 10-3) hits, of which 698 (42.5%) were annotated with Gene Ontology terms (E-value < 10-6). The number of unknown unique sequences was 946 (57.5%). All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021). Gene discovery and annotations are presented and discussed.ConclusionThis set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.

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