High-throughput verification of transcriptional starting sites by Deep-RACE
ABSTRACT 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.
Full-textDOI: · Available from: Piero Carninci, Jul 29, 2015
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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. "
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 · 3.71 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. "
ABSTRACT: The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5′-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called “Deep-RACE”, which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3′ region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes. Electronic supplementary material The online version of this article (doi:10.1007/s00439-013-1356-6) contains supplementary material, which is available to authorized users.Human Genetics 09/2013; 133(1). DOI:10.1007/s00439-013-1356-6 · 4.52 Impact Factor
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- "The TSSs detected do not occur isolated but are arranged in regions generally referred to as a TSS cluster with the core promoter region loosely defined as the genomic region surrounding it (Carninci et al. 2006; Sandelin et al. 2007). To this purpose, we developed our own specific protocol , related to previous methods (Olivarius et al. 2009; Tsuchihara et al. 2009), which we dubbed 59-MACE (massive amplification of cDNA ends). Shortly, we combined RNA oligo-capping with reverse transcription using tagged random hexamers to generate a cDNA Figure 2. Expression levels of PcG and TrxG target genes. "
ABSTRACT: The Polycomb group (PcG) and Trithorax group (TrxG) of proteins are required for stable and heritable maintenance of repressed and active gene expression states. Their antagonistic function on gene control, repression for PcG and activity for TrxG, is mediated by binding to chromatin and subsequent epigenetic modification of target loci. Despite our broad knowledge about composition and enzymatic activities of the protein complexes involved, our understanding still lacks important mechanistic detail and a comprehensive view on target genes. In this study we use an extensive data set of ChIP-seq, RNA-seq, and genome-wide detection of transcription start sites (TSSs) to identify and analyze thousands of binding sites for the PcG proteins and Trithorax from a Drosophila S2 cell line. In addition of finding a preference for stalled promoter regions of annotated genes, we uncover many intergenic PcG binding sites coinciding with nonannotated TSSs. Interestingly, this set includes previously unknown promoters for primary transcripts of microRNA genes, thereby expanding the scope of Polycomb control to noncoding RNAs essential for development, apoptosis, and growth.Genome Research 02/2011; 21(2):216-26. DOI:10.1101/gr.114348.110 · 13.85 Impact Factor