A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.


BACKGROUND:cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz. RESULTS:In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development. CONCLUSION:We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.


BMC Mol Biol


BMC molecular biology


Kuvbachieva AA,Goffinet AM





Has Abstract


2002-04-24 00:00:00








  • From Gateway to MultiSite Gateway in one recombination event.

    abstract:BACKGROUND:Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gat...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Magnani E,Bartling L,Hake S

    更新日期:2006-12-06 00:00:00

  • GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells.

    abstract:BACKGROUND:We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Di Lisi R,Picard A,Ausoni S,Schiaffino S

    更新日期:2007-09-17 00:00:00

  • Characterization, phylogeny, alternative splicing and expression of Sox30 gene.

    abstract:BACKGROUND:Members of the Sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. Among these members, Sox30 had been considered to exist only in mammals since its discovery, and its e...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Han F,Wang Z,Wu F,Liu Z,Huang B,Wang D

    更新日期:2010-12-11 00:00:00

  • RE1 silencing transcription factor (REST) negatively regulates ALL1-fused from chromosome 1q (AF1q) gene transcription.

    abstract:BACKGROUND:ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive. RESULTS:Our study showed that REST (RE1 silencing transcription factor), a key transcription factor ...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Hu Y,Sun Q,Zhang C,Sha Q,Sun X

    更新日期:2015-09-05 00:00:00

  • cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene.

    abstract:BACKGROUND:The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in re...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Thiel G,Al Sarraj J,Stefano L

    更新日期:2005-01-19 00:00:00

  • Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR.

    abstract:BACKGROUND:The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amp...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Rutledge RG,Stewart D

    更新日期:2008-10-30 00:00:00

  • The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase.

    abstract:BACKGROUND:Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interes...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Purta E,van Vliet F,Tkaczuk KL,Dunin-Horkawicz S,Mori H,Droogmans L,Bujnicki JM

    更新日期:2006-07-18 00:00:00

  • Novel insight into the composition of human single-stranded DNA-binding protein 1 (hSSB1)-containing protein complexes.

    abstract:BACKGROUND:Single-stranded DNA-binding proteins are essential cellular components required for the protection, metabolism and processing of single-stranded DNA. Human single-stranded DNA-binding protein 1 (hSSB1) is one such protein, with described roles in genome stability maintenance and transcriptional regulation. A...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Ashton NW,Loo D,Paquet N,O'Byrne KJ,Richard DJ

    更新日期:2016-12-09 00:00:00

  • Alternative splicing and differential subcellular localization of the rat FGF antisense gene product.

    abstract:BACKGROUND:GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Zhang SC,MacDonald KA,Baguma-Nibasheka M,Geldenhuys L,Casson AG,Murphy PR

    更新日期:2008-01-23 00:00:00

  • Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase.

    abstract:BACKGROUND:Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical proper...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Kim YG,Yoo JS,Kim JH,Kim CM,Oh JW

    更新日期:2007-07-11 00:00:00

  • Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae.

    abstract:BACKGROUND:The human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two reg...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Yousef AF,Brandl CJ,Mymryk JS

    更新日期:2009-04-17 00:00:00

  • Structural and functional analysis of four non-coding Y RNAs from Chinese hamster cells: identification, molecular dynamics simulations and DNA replication initiation assays.

    abstract:BACKGROUND:The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect repli...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: de Lima Neto QA,Duarte Junior FF,Bueno PS,Seixas FA,Kowalski MP,Kheir E,Krude T,Fernandez MA

    更新日期:2016-01-05 00:00:00

  • Relative transcript quantification by quantitative PCR: roughly right or precisely wrong?

    abstract:BACKGROUND:When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS:Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, di...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Skern R,Frost P,Nilsen F

    更新日期:2005-04-26 00:00:00

  • A small intergenic region drives exclusive tissue-specific expression of the adjacent genes in Arabidopsis thaliana.

    abstract:BACKGROUND:Transcription initiation by RNA polymerase II is unidirectional from most genes. In plants, divergent genes, defined as non-overlapping genes organized head-to-head, are highly represented in the Arabidopsis genome. Nevertheless, there is scarce evidence on functional analyses of these intergenic regions. Th...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Bondino HG,Valle EM

    更新日期:2009-10-16 00:00:00

  • New insights into SRY regulation through identification of 5' conserved sequences.

    abstract:BACKGROUND:SRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones. RESULTS:We identified four intervals of high homology upstr...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Ross DG,Bowles J,Koopman P,Lehnert S

    更新日期:2008-10-14 00:00:00

  • Transcriptional response of pancreatic beta cells to metabolic stimulation: large scale identification of immediate-early and secondary response genes.

    abstract:BACKGROUND:Physiological long term adaptation of pancreatic beta cells is driven by stimuli such as glucose and incretin hormones acting via cAMP (e.g. GLP-1) and involves regulated gene expression. Several rapidly inducible immediate-early genes (IEGs) have been identified in beta cells. Many of these IEGs code for tr...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Glauser DA,Brun T,Gauthier BR,Schlegel W

    更新日期:2007-06-22 00:00:00

  • RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX.

    abstract:BACKGROUND:Modulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processe...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Pinato S,Scandiuzzi C,Arnaudo N,Citterio E,Gaudino G,Penengo L

    更新日期:2009-06-05 00:00:00

  • Sequence periodicity of Escherichia coli is concentrated in intergenic regions.

    abstract:BACKGROUND:Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli sequences display the period close to 11 base pairs. RESULTS:Here we demonstrate that practically only ApA/TpT dinucle...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Hosid S,Trifonov EN,Bolshoy A

    更新日期:2004-08-26 00:00:00

  • Single and combinatorial chromatin coupling events underlies the function of transcript factor Krüppel-like factor 11 in the regulation of gene networks.

    abstract:BACKGROUND:Krüppel-like factors (KLFs) are a group of master regulators of gene expression conserved from flies to human. However, scant information is available on either the mechanisms or functional impact of the coupling of KLF proteins to chromatin remodeling machines, a deterministic step in transcriptional regula...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Calvo E,Grzenda A,Lomberk G,Mathison A,Iovanna J,Urrutia R

    更新日期:2014-05-25 00:00:00

  • Alternative splicing of the neurofibromatosis type 1 pre-mRNA is regulated by the muscleblind-like proteins and the CUG-BP and ELAV-like factors.

    abstract:BACKGROUND:Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. One major challenge is to identify all of the protein players and define how they control alternative expression of a particular exon in a combinatorial manner. The M...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Fleming VA,Geng C,Ladd AN,Lou H

    更新日期:2012-12-10 00:00:00

  • Discriminating nucleosomes containing histone H2A.Z or H2A based on genetic and epigenetic information.

    abstract:BACKGROUND:Nucleosomes are nucleoproteic complexes, formed of eight histone molecules and DNA, and they are responsible for the compaction of the eukaryotic genome. Their presence on DNA influences many cellular processes, such as transcription, DNA replication, and DNA repair. The evolutionarily conserved histone vari...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Gervais AL,Gaudreau L

    更新日期:2009-03-04 00:00:00

  • A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity.

    abstract:BACKGROUND:Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purificatio...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Voss C,Schmitt B,Werner-Simon S,Lutz C,Simon W,Anderl J

    更新日期:2014-04-03 00:00:00

  • Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate.

    abstract:BACKGROUND:The human Sex Hormone Binding Globulin (SHBG) gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the ...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Pinós T,Barbosa-Desongles A,Hurtado A,Santamaria-Martínez A,de Torres I,Morote J,Reventós J,Munell F

    更新日期:2009-06-17 00:00:00

  • Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis.

    abstract:BACKGROUND:Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis. R...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: He H,Cheng M,Yang X,Meng J,He A,Zheng X,Li Z,Guo P,Pan Z,Zhan X

    更新日期:2009-10-25 00:00:00

  • A reliable method for quantification of splice variants using RT-qPCR.

    abstract:BACKGROUND:The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use. RESU...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Camacho Londoño J,Philipp SE

    更新日期:2016-03-15 00:00:00

  • Rad9 modulates the P21WAF1 pathway by direct association with p53.

    abstract:BACKGROUND:Previous studies suggest that human RAD9 (hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival P21WAF1. This study exam...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Ishikawa K,Ishii H,Murakumo Y,Mimori K,Kobayashi M,Yamamoto K,Mori M,Nishino H,Furukawa Y,Ichimura K

    更新日期:2007-05-21 00:00:00

  • Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis.

    abstract:BACKGROUND:In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs). The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regu...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Jin H,Vacic V,Girke T,Lonardi S,Zhu JK

    更新日期:2008-01-14 00:00:00

  • Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells.

    abstract:BACKGROUND:Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated ...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Li T,Diao H,Zhao L,Xing Y,Zhang J,Liu N,Yan Y,Tian X,Sun W,Liu B

    更新日期:2017-04-05 00:00:00

  • Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1.

    abstract:BACKGROUND:Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acc...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Zaniolo K,Desnoyers S,Leclerc S,Guérin SL

    更新日期:2007-10-25 00:00:00

  • Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter.

    abstract:BACKGROUND:Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skelet...

    journal_title:BMC molecular biology

    pub_type: 杂志文章


    authors: Ounzain S,Dacwag CS,Samani NJ,Imbalzano AN,Chong NW

    更新日期:2008-05-19 00:00:00