BMC Molecular Biology最新文献

{"title":"MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA","authors":"Yi Jin,&nbsp;Fang Zhang,&nbsp;Zhenfa Ma,&nbsp;Zhuqing Ren","doi":"10.1186/s12867-016-0070-z","DOIUrl":"https://doi.org/10.1186/s12867-016-0070-z","url":null,"abstract":"<p>Nonsense-mediated mRNA decay (NMD) is a RNA quality surveillance system for eukaryotes. It prevents cells from generating deleterious truncated proteins by degrading abnormal mRNAs that harbor premature termination codon (PTC). However, little is known about the molecular regulation mechanism underlying the inhibition of NMD by microRNAs.</p><p>The present study demonstrated that miR-433 was involved in NMD pathway via negatively regulating <i>SMG5</i>. We provided evidence that (1) overexpression of miR-433 significantly suppressed the expression of <i>SMG5</i> (P?&lt;?0.05); (2) Both mRNA and protein expression levels of <i>TBL2</i> and <i>GADD45B</i>, substrates of NMD, were increased when <i>SMG5</i> was suppressed by siRNA; (3) Expression of <i>SMG5</i>, <i>TBL2</i> and <i>GADD45B</i> were significantly increased by miR-433 inhibitor (P?&lt;?0.05). These results together illustrated that miR-433 regulated NMD by targeting <i>SMG5</i> mRNA.</p><p>Our study highlights that miR-433 represses nonsense mediated mRNA decay. The miR-433 targets 3’-UTR of <i>SMG5</i> and represses the expression of <i>SMG5</i>, whereas NMD activity is decreased when SMG5 is decreased. This discovery provides evidence for microRNA/NMD regulatory mechanism.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0070-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5117812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stearoyl-CoA desaturase 1 expression is downregulated in liver and udder during E. coli mastitis through enhanced expression of repressive C/EBP factors and reduced expression of the inducer SREBP1A","authors":"Tianle Xu,&nbsp;Xiangzhen Shen,&nbsp;Hans-Martin Seyfert","doi":"10.1186/s12867-016-0069-5","DOIUrl":"https://doi.org/10.1186/s12867-016-0069-5","url":null,"abstract":"<p>Stearoyl-CoA desaturase 1 (SCD1) desaturates long chain fatty acids and is therefore a key enzyme in fat catabolism. Its synthesis is downregulated in liver during illnesses caused by high levels of circulating lipopolysaccharide (LPS). SCD1 expression is known to be stimulated under adipogenic conditions through a variety of transcription factors, notably SREBP1 and C/EBPα and ?β. However, mechanisms downregulating SCD1 expression during illness related reprograming of the metabolism were unknown. <i>Escherichia coli</i> elicited mastitis is an example of such a condition and was found to downregulates milk and milk fat synthesis. This is in part mediated through epigenetic mechanisms. We analyzed here mechanism controlling SCD1 expression in livers and udders from cows suffering from experimentally induced <i>E. coli</i> mastitis.</p><p>We validated with RT-qPCR that SCD1 expression was reduced in these organs of the experimental cows. They also featured decreased levels of mRNAs encoding SREBP1a but increased levels for C/EBP α and ?β. Chromatin accessibility PCR (CHART) revealed that downregulation of SCD1 expression in liver was not caused by tighter chromatin compaction of the SCD1 promoter. Reporter gene analyses showed in liver (HepG2) and mammary epithelial (MAC-T) model cells that overexpression of SREBP1a expectedly activated the promoter, while unexpectedly C/EBPα and ?β strongly quenched the promoter activity. Abrogation of two from among of the three C/EBP DNA-binding motifs of the promoter revealed that C/EBPα acts in <i>cis</i> but C/EBPβ in <i>trans</i>. Overexpressing truncated C/EBPα or ?β factors lacking their repressive domains confirmed in both model cells the direct action of C/EBPα, but not of C/EBPβ on the promoter.</p><p>We found no evidence that epigenetic mechanism remodeling the chromatin compaction of the SCD1 promoter would contribute to downregulate SCD1 expression during infection. Instead, our data show for the first time that C/EBP factors may repress SCD1 expression in liver and udder rather than stimulating as it was previously shown in adipocytes. This cell type specific dual and opposite function of C/EBP factors for regulating SCD1 expression was previously unknown. Infection related activation of their expression combined with downregulated expression of SREBP1a explains reduced SCD1 expression in liver and udder during acute mastitis.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0069-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4790993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein","authors":"Victoria L. M. Herrera,&nbsp;Martin Steffen,&nbsp;Ann Marie Moran,&nbsp;Glaiza A. Tan,&nbsp;Khristine A. Pasion,&nbsp;Keith Rivera,&nbsp;Darryl J. Pappin,&nbsp;Nelson Ruiz-Opazo","doi":"10.1186/s12867-016-0066-8","DOIUrl":"https://doi.org/10.1186/s12867-016-0066-8","url":null,"abstract":"<p>In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly <i>Dear</i>) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein’s existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions.</p><p>To dissect the nucleotide sequence discrepancy, we performed Maxam–Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR’s existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects.</p><p>Maxam–Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5?kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14.</p><p>Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0066-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4870908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strong preference of BRCA1 protein to topologically constrained non-B DNA structures","authors":"Václav Brázda,&nbsp;Lucia Hároníková,&nbsp;Jack C. C. Liao,&nbsp;Helena Fridrichová,&nbsp;Eva B. Jagelská","doi":"10.1186/s12867-016-0068-6","DOIUrl":"https://doi.org/10.1186/s12867-016-0068-6","url":null,"abstract":"<p>The breast and ovarian cancer susceptibility gene <i>BRCA1</i> encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail.</p><p>In this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444–1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy.</p><p>Our discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0068-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4343678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum to: Gene expression profiling of the venom gland from the Venezuelan mapanare (Bothrops colombiensis) using expressed sequence tags (ESTs)","authors":"Montamas Suntravat,&nbsp;Néstor L. Uzcategui,&nbsp;Chairat Atphaisit,&nbsp;Thomas J. Helmke,&nbsp;Sara E. Lucena,&nbsp;Elda E. Sánchez,&nbsp;Alexis Rodríguez-Acosta","doi":"10.1186/s12867-016-0062-z","DOIUrl":"https://doi.org/10.1186/s12867-016-0062-z","url":null,"abstract":"","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0062-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4943007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A potential role for protein palmitoylation and zDHHC16 in DNA damage response","authors":"Na Cao,&nbsp;Jia-Kai Li,&nbsp;Yu-Qing Rao,&nbsp;Huijuan Liu,&nbsp;Ji Wu,&nbsp;Baojie Li,&nbsp;Peiquan Zhao,&nbsp;Li Zeng,&nbsp;Jing Li","doi":"10.1186/s12867-016-0065-9","DOIUrl":"https://doi.org/10.1186/s12867-016-0065-9","url":null,"abstract":"<p>Cells respond to DNA damage by activating the phosphatidylinositol-3 kinase-related kinases, p53 and other pathways to promote cell cycle arrest, apoptosis, and/or DNA repair. Here we report that protein palmitoylation, a modification carried out by protein acyltransferases with zinc-finger and Asp-His-His-Cys domains (zDHHC), is required for proper DNA damage responses.</p><p>Inhibition of protein palmitoylation compromised DNA damage-induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts. Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c-Abl, a non-receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation.</p><p>Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0065-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4431090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mice lacking microRNAs in Pax8-expressing cells develop hypothyroidism and end-stage renal failure","authors":"Malte P. Bartram,&nbsp;Elena Amendola,&nbsp;Thomas Benzing,&nbsp;Bernhard Schermer,&nbsp;Gabriella de Vita,&nbsp;Roman-Ulrich Müller","doi":"10.1186/s12867-016-0064-x","DOIUrl":"https://doi.org/10.1186/s12867-016-0064-x","url":null,"abstract":"<p>Non-coding RNAs have gained increasing attention during the last decade. The first large group of non-coding RNAs to be characterized systematically starting at the beginning of the 21st century were small oligonucleotides—the so-called microRNAs (miRNAs). By now we have learnt that microRNAs are indispensable for most biological processes including organogenesis and maintenance of organ structure and function. The role of microRNAs has been studied extensively in the development of a number of organs, so far most studies focussed on e.g. the heart or the brain whilst the role of microRNAs in the development and maintenance of complex epithelial organs is less well understood. Furthermore most analyses regarding microRNA function in epithelial organs employed conditional knockout mouse models of the RNAse III Dicer to abrogate microRNA biogenesis. However, there is increasing evidence for Dicer to have multiple functions independent from microRNA maturation. Therefore Dicer independent models are needed to gain further insight into the complex biology of miRNA dependent processes.</p><p>Here we analyze the contribution of microRNA-dependent transcriptional control in Pax8-expressing epithelial cells. Pax8 is a transcription factor that is crucial to the development of epithelial organs. The miRNA machinery was disrupted by crossing conditional <i>DiGeorge syndrome critical region 8</i> \u0000 <i>(Dgcr8) fl/fl</i> mice to <i>Pax8Cre</i> mice. The Dgcr8/Drosha complex processes pri-miRNAs in the nucleus before they are exported as pre-miRNAs for further maturation by Dicer in the cytoplasm. <i>Dgcr8</i>?<i>fl/fl; Pax8Cre</i>+?knockout mice died prematurely, developed massive hypothyroidism and end stage renal disease due to a loss of miRNAs in Pax8 expressing tissue.</p><p>\u0000 <i>Pax8Cre</i>-mediated conditional loss of DiGeorge syndrome critical region 8 (Dgcr8), an essential component of the nuclear machinery that is required for microRNA biogenesis, resulted in severe hypothyroidism, massively reduced body weight and ultimately led to renal failure and death of the animals. These data provide further insight into the importance of miRNAs in organ homeostasis using a Dicer independent model.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0064-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4708816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer","authors":"Sophia Pinz,&nbsp;Samy Unser,&nbsp;Anne Rascle","doi":"10.1186/s12867-016-0063-y","DOIUrl":"https://doi.org/10.1186/s12867-016-0063-y","url":null,"abstract":"<p>\u0000 <i>c</i>-<i>Myc</i> has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the <i>c</i>-<i>Myc</i> gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. <i>c</i>-<i>Myc</i> super-enhancer, located 1.7?Mb downstream of the <i>c</i>-<i>Myc</i> gene locus, was recently reported as essential for the regulation of <i>c</i>-<i>Myc</i> gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. <i>c</i>-<i>Myc</i> super-enhancer is composed of five regulatory regions (E1–E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the <i>c</i>-<i>Myc</i> promoter.</p><p>We now show that STAT5 strongly binds to <i>c</i>-<i>Myc</i> super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with <i>c</i>-<i>Myc</i> transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and <i>c</i>-<i>Myc</i> gene expression.</p><p>Our data suggest that constitutive STAT5 binding to <i>c</i>-<i>Myc</i> super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of <i>c</i>-<i>Myc</i> in Ba/F3 cells transformed by STAT5-1*6.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0063-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4560796","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection","authors":"Jason A. Estep,&nbsp;Erin L. Sternburg,&nbsp;Gissell A. Sanchez,&nbsp;Fedor V. Karginov","doi":"10.1186/s12867-016-0061-0","DOIUrl":"https://doi.org/10.1186/s12867-016-0061-0","url":null,"abstract":"<p>Targeted genomic editing using the CRISPR/Cas9 methodology has opened exciting new avenues in probing gene function in virtually any model system, including cultured mammalian cells. Depending on the desired mutation, several experimental options exist in the isolation of clonal lines, such as selection with introduced markers, or screening by PCR amplification of genomic DNA. However, streamlined approaches to establishing deletion and tagging mutants with minimal genomic perturbation are of interest in applying this methodology.</p><p>We developed a procedure for rapid screening of clonal cell lines for the deletion of a protein of interest following CRISPR/Cas9 targeting in the absence of selective pressure based on dot immunoblots. To assess the technique, we probed clonal isolates of 293-TREx cells that were targeted with three separate sgRNAs against the HuR gene. Validation of knockout candidates by western blot indicated that the normalized protein abundances indicated by the dot blot serve as accurate predictors of deletion. In total, 32 independent biallelic deletion lines out of 248 screened clones were isolated, and recovery of null mutants ranged from 6?to 36?% for the individual sgRNAs. Genomic sequencing verified small deletions at the targeted locus.</p><p>Clonal screening for CRISPR/Cas9-mediated editing events using dot immunoblot is a straightforward and efficient approach that facilitates rapid generation of genomic mutants to study gene function.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0061-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4063568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A reliable method for quantification of splice variants using RT-qPCR","authors":"Julia Camacho Londoño,&nbsp;Stephan E. Philipp","doi":"10.1186/s12867-016-0060-1","DOIUrl":"https://doi.org/10.1186/s12867-016-0060-1","url":null,"abstract":"<p>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.</p><p>Here, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified within a single sample using one-step reverse transcription quantitative PCR. Amplification products obtained with variant specific primer pairs were compared to those obtained with primer pairs common to both variants. The identities of variant specific amplicons were simultaneously verified by melt curve analysis. Independent calculations of the relative incidence of each variant were performed. Since the relative incidences of variants have to add upto 100?%, the method provides an internal control to monitor experimental errors and uniform reverse transcription. The reliability of the method was tested using mixtures of cDNA templates as well as RNA samples from different sources.</p><p>The method described here, is easy to set up and does not need unrelated reference genes and time consuming, error-prone standard curves. It provides a reliable and precise technique to distinguish small differences of the relative incidence of two splice variants.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"17 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2016-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-016-0060-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4619556","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}