Normalization and Subtraction of Cap-Trapper-Selected cDNAs to Prepare Full-Length cDNA Libraries for Rapid Discovery of New Genes

Carninci, Piero; Shibata, Yûkô; Hayatsu, Norihito; Sugahara, Yuichi; Shibata, Kazuhiro; Itoh, Masayoshi; Konno, Hiroyuki; Okazaki, Yasushi; Muramatsu, Masami; Hayashizaki, Yoshihide

doi:10.1101/gr.145100

Cited by 264 publications

(190 citation statements)

References 22 publications

(31 reference statements)

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“…During reverse transcription, adaptors containing the asymmetrical restriction sites for SfiI were incorporated into the first strand of the cDNA using a SMART template-switching mechanism at the 5Ј-end of the transcript (47). To decrease redundancy, all libraries were normalized by hybridization of the single-strand cDNA with the same pool of mRNA that was used for first-strand synthesis (9). Second-strand synthesis of cDNA was performed by the use of primer extension PCR (Advantage 2 Taq Polymerase, Clontech) with limited (10 -15) number of cycles.…”

Section: Methodsmentioning

confidence: 99%

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Fedorov

Goropashnaya

Tøien

et al. 2009

Physiological Genomics

125

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We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver. gene expression; hibernation; translation; RNA binding protein motif 3 MAMMALIAN HIBERNATION IS a physiological and behavioral adaptation involving a coordinated suppression of heat production and body temperature wherein whole body metabolism and energy demand are significantly reduced over several days to several months. During hibernation, heart and respiration rates, blood flow, and oxygen consumption decrease dramatically to Ͻ10% of normal or basal rates (2, 4). These physiological changes do not represent a loss of homeostasis but instead are precisely controlled and spontaneously reversible, and they allow individual animals to survive highly seasonal or unpredictable environments where food availability becomes lacking (7, 12). The molecular and genetic basis of hibernation in small mammals has only recently begun to be described, and little is known about its evolutionary history. Hibernating species have been found in diverse families among seven orders of mammals (26), however, and the interspersed phylogenetic distribution of hibernating and nonhibernating species has led to the hypothesis that rather than requiring the creation of novel gene products, hibernation results from the differential expression of genes that exist widely among mammals (35).Microarray technology provides powerful means for the unbiased detection of differences in expression of thousands of genes in a single hybridization experiment (14). In contrast to single-gene expression analysis, the genome-wide approach also allows for identification of coordinated transcriptional changes in functional groups of regulatory genes within metabolic and signaling pathways. Recent studies of differential gen...

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Section: Methodsmentioning

confidence: 99%

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Fedorov

Goropashnaya

Tøien

et al. 2009

Physiological Genomics

125

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“…The cDNA clones used in the project were selected from 246 fulllength enriched cDNA libraries, most of which were also normalized and subtracted, with most from C57BL/6J mice, as described elsewhere 12,14,15 . Information about tissue source and other information regarding these libraries is available in Supplementary Information section 1.…”

Section: The Fantom2 Clone Setmentioning

confidence: 99%

“…1,442,236 sequences were grouped into 171,144 3 0 -end clusters based on sequence similarity (Supplementary Information section 2) [14][15][16] . Of these, 159,789 had no significant BLAST hit to known mouse genes, and were considered potentially novel.…”

Section: The Fantom2 Clone Setmentioning

confidence: 99%

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Okazaki¹,

Furuno²,

Kasukawa³

et al. 2002

Nature

1,451

406

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Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

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“…In the present study, we printed arrays with preeclampsia-associated cDNAs obtained from subtracted libraries (23), and compared gene expression in placenta samples from women with PE, bilateral notching, PE plus bilateral notching and healthy controls. Our goal in doing this was to look for alterations in genes that might contribute to or protect the placenta from PE.…”

Section: Introductionmentioning

confidence: 99%

Placental expression profiling in preeclampsia: local overproduction of hemoglobin may drive pathological changes

Centlow

Carninci²,

Nemeth

et al. 2008

Fertility and Sterility

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Using microarrays made with cDNAs from subtracted placental libraries, we show increased hemoglobin production in the preeclamptic placenta. Heme and hemoglobin may cause endothelial damage and inflammation and drive pathological changes in the placenta if they are released there. Study Objective:To create a library enriched in cDNAs from preeclamptic placentas to print on to microarrays for placental profiling of preeclampsia (PE) and high risk pregnancies. Design: Prospective study.Setting: University women's clinic and academic research laboratory.Patients: Ten patients with PE, 5 with PE and bilateral notching, 5 with bilateral notching without PE, and 15 normotensive patients were recruited.Interventions: Placenta and placenta bed biopsies were collected after delivery. Main Outcome Measures:Subtracted libraries of PE transcripts were produced, and cDNAs from these libraries were used to make PE-specific cDNA arrays. Results were verified quantitatively using real-time PCR and histologically using in situ hybridization and immunohistochemistry.Results: 30 genes were significantly altered in at least one group comparison. Differences in two candidate genes were confirmed using quantitative rt-PCR. Hemoglobin α2 and γ transcripts were significantly over-expressed in the PE placenta. Scattered cells in the placenta and placental blood vessels were shown to express genes encoding these hemoglobin-chains. Conclusions:We demonstrate increased hemoglobin production in the PE placenta. The hemoglobin may be released into the placenta blood vessel lumen. Free heme and hemoglobin are potent toxins which cause endothelial damage and inflammation.

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Normalization and Subtraction of Cap-Trapper-Selected cDNAs to Prepare Full-Length cDNA Libraries for Rapid Discovery of New Genes

Cited by 264 publications

References 22 publications

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Placental expression profiling in preeclampsia: local overproduction of hemoglobin may drive pathological changes

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