Full-Length Transcriptome Analysis Using a Bias-Free cDNA Library Prepared with the Vector-Capping Method

Kato, Souichiro; Oshikawa, Mio; Ohtoko, Kuniyo

doi:10.1007/978-1-61779-065-2_4

Cited by 3 publications

(5 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have constructed 32 cDNA libraries for 28 different tissues and cell populations by using cloning of cap-structured mRNA [20,21] or the SMART method [22], and we have accumulated 330,707 ESTs from 5′-ends (Table 1) including previously reported 162,631 ESTs in our pig expressed gene database [18,19]. The ESTs thus obtained were assembled into 17,183 contigs consisting of 209,779 ESTs, with 120,928 singlets remaining.…”

Section: Resultsmentioning

confidence: 99%

“…Twenty-three libraries were constructed by using the oligo-capping method [21], and five were constructed by using the vector-capping [20] method. Four libraries were constructed by using the SMART method (Clontech, Palo Alto, CA, USA) [22].…”

Section: Resultsmentioning

confidence: 99%

“…Fifteen of the oligo-capped cDNA libraries were constructed by using Gateway-compatible pCMVFL3 vector (Invitrogen, Carlsbad, CA, USA) [53], whereas the vector for the rest was pME18SFL3 (Toyobo, Osaka, Japan). Five cDNA libraries were constructed by using another method for constructing full-length cDNA libraries, namely the vector-capping method [20]. In total, 28 libraries were generated by methods using the 5′ cap structure, which is characteristic of intact mRNA.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

Uenishi

Morozumi²,

Toki³

et al. 2012

BMC Genomics

View full text Add to dashboard Cite

BackgroundAlong with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits.ResultsWe performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5′-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome.ConclusionsESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

Uenishi

Morozumi²,

Toki³

et al. 2012

BMC Genomics

View full text Add to dashboard Cite

show abstract

“…A plasmid harbouring the FLNC open reading frame (ORF) (ARi57A02) was provided by RIKEN BioResource Center (Tsukuba, Japan) through the National Bio-Resource, Japan. 31–33 FLNC and enhanced green fluorescent protein (EGFP) ORFs were ligated into pCDNA3.1(+) (Thermo Fisher Scientific), and pCDNA3.1(+)-FLAG-FLNC and pCDNA3.1(+)-FLAG-EGFP were transfected into LN229 and U251MG cell lines using Lipofectamine 2000 (Thermo Fisher Scientific). Transfected cells were inoculated in a 10-cm 2 dish (1000 cells/dish) for cloning.…”

Section: Methodsmentioning

confidence: 99%

High filamin-C expression predicts enhanced invasiveness and poor outcome in glioblastoma multiforme

et al. 2019

View full text Add to dashboard Cite

Background Glioblastoma multiforme (GBM), the most common brain malignancy in adults, is generally aggressive and incurable, even with multiple treatment modalities and agents. Filamins (FLNs) are a group of actin-binding proteins that regulate the actin cytoskeleton in cells. However, the role of FLNs in malignancies—particularly in GBM—is unclear. Methods The relation between FLNC expression and overall survival in GBM was evaluated by the Kaplan−Meier analysis using GBM patients from the Kagoshima University Hospital ( n = 90) and data from the Cancer Genome Atlas (TCGA) ( n = 153). To assess FLNC function in GBM, cell migration and invasion were examined with Transwell and Matrigel invasion assays using FLNC-overexpressing U251MG and LN299 GBM cells, and ShRNA-mediated FLNC knocked-down KNS81 and U87MG cells. The gelatin zymography assay was used to estimate matrix metalloproteinase (MMP) 2 activity. Results In silico analysis of GBM patient data from TCGA and immunohistochemical analyses of clinical GBM specimens revealed that increased FLNC expression was associated with poor patient prognosis. FLNC overexpression in GBM cell lines was positively correlated with enhanced invasiveness, but not migration, and was accompanied by upregulation of MMP2. Conclusions FLNC is a potential therapeutic target and biomarker for GBM progression.

show abstract

“…Based on the most recent statistics Full-length cDNA sequences are more valuable than fragmented ESTs to fully understand the fine structures, transcript diversity in pig transcriptome, and for functional studies. Full-length cDNA enriched libraries were constructed by using oligo-capping [30], vector-capping [31], SMART [32,33] or more recently-developed TeloPrime [34] methods. To date, full-length cDNA sequences are available for 28 tissues [29].…”

Section: Sanger Method-based Est and Full-length Cdna Sequencingmentioning

confidence: 99%

Swine blood transcriptomics: Application and advancement

Li¹

View full text Add to dashboard Cite

Full-Length Transcriptome Analysis Using a Bias-Free cDNA Library Prepared with the Vector-Capping Method

Cited by 3 publications

References 9 publications

Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

High filamin-C expression predicts enhanced invasiveness and poor outcome in glioblastoma multiforme

Swine blood transcriptomics: Application and advancement

Contact Info

Product

Resources

About