High-throughput verification of transcriptional starting sites by Deep-RACE

Functional Genomics Technology Team, Omics Science Center, RIKEN Yokohama Institute, Kanagawa, Japan.
BioTechniques (Impact Factor: 2.95). 03/2009; 46(2):130-2. DOI: 10.2144/000113066
Source: PubMed


We present a high-throughput method for investigating the transcriptional starting sites of genes of interest, which we named Deep-RACE (Deep-rapid amplification of cDNA ends). Taking advantage of the latest sequencing technology, it allows the parallel analysis of multiple genes and is free of time-consuming cloning steps. In comparison to the sequencing of RACE PCR products, our approach is more precise and more cost-effective even for batches as small as 17.

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    • "3 0 -RACE-PCR was performed using UPM together with an LvCTL3-specific forward primer 3RACE1, and the nested PCR was subsequently performed with NUP and LvCTL3–3RACE2. The second PCR products were cloned into pMD-20T vector (TaKaRa, Japan) and 12 positive clones were selected and sequenced (ABI PRISM, Applied Biosystems, USA). 5 0 -RACE is a technique used in molecular biology to obtain the 5 0 UTR of a transcript and identify the transcription starting site (TSS) of promoter elements (Olivarius et al., 2009; Scotto-Lavino et al., 2006). TSS of LvCTL3 is determined according to the 5 0 -RACE PCR amplification. "
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    ABSTRACT: C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated 'EPD' motif in Ca(2+)-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5' flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-Luciferase Reporter Assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could been regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but can not agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca(2+) was chelated with EDTA, suggesting the function of LvCTL3 is Ca(2+)-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection.
    Developmental and comparative immunology 04/2014; 46(2). DOI:10.1016/j.dci.2014.04.014 · 2.82 Impact Factor
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    • "In order to overcome these limitations, refinements of NGS-based methods have been developed to map TSS and PAS. One approach involves the use of tobacco acid pyrophosphatase (TAP) to remove the 5′ cap and allow ligation of a sequencing adapter specifically to the 5′-end of the RNA (10–12). To map PAS, methods based on oligo(dT) priming or poly(A) capture have been used (13–19). "
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    ABSTRACT: Understanding the relationships between regulatory factor binding, chromatin structure, cis-regulatory elements and RNA-regulation mechanisms relies on accurate information about transcription start sites (TSS) and polyadenylation sites (PAS). Although several approaches have identified transcript ends in yeast, limitations of resolution and coverage have remained, and definitive identification of TSS and PAS with single-nucleotide resolution has not yet been achieved. We developed SMORE-seq (simultaneous mapping of RNA ends by sequencing) and used it to simultaneously identify the strongest TSS for 5207 (90%) genes and PAS for 5277 (91%) genes. The new transcript annotations identified by SMORE-seq showed improved distance relationships with TATA-like regulatory elements, nucleosome positions and active RNA polymerase. We found 150 genes whose TSS were downstream of the annotated start codon, and additional analysis of evolutionary conservation and ribosome footprinting suggests that these protein-coding sequences are likely to be mis-annotated. SMORE-seq detected short non-coding RNAs transcribed divergently from more than a thousand promoters in wild-type cells under normal conditions. These divergent non-coding RNAs were less evident at promoters containing canonical TATA boxes, suggesting a model where transcription initiation at promoters by RNAPII is bidirectional, with TATA elements serving to constrain the directionality of initiation.
    Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1366 · 9.11 Impact Factor
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    • "In this study we used an innovative approach called Deep-RACE, which combines RACE and next generation sequencing (Olivarius et al. 2009) to identify novel transcripts in high-throughput manner. This technique is highly sensitive and enables detection of very low abundance transcripts. "
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