Abstract:
:Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Baugh LR,Hill AA,Brown EL,Hunter CPdoi
10.1093/nar/29.5.e29keywords:
subject
Has Abstractpub_date
2001-03-01 00:00:00pages
E29issue
5eissn
0305-1048issn
1362-4962journal_volume
29pub_type
杂志文章abstract::P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53B...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz138
更新日期:2019-04-08 00:00:00
abstract::A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
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doi:10.1093/nar/11.15.5093
更新日期:1983-08-11 00:00:00
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journal_title:Nucleic acids research
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更新日期:2013-02-01 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:1991-06-11 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:1977-07-01 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2008-11-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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abstract::We present YOGY a web-based resource for orthologous proteins from nine eukaryotic organisms: Homo sapiens, Mus musculus, Rattus norvegicus, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, Plasmodium falciparum, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Using a gene name from any o...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2019-01-08 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2009-04-01 00:00:00
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更新日期:1997-07-15 00:00:00