Oligonucleotides used as template calibrators for general application in quantitative polymerase chain reaction.
ABSTRACT The optimizing and controlling for polymerase chain reactions (PCRs) requires standard target sequences to measure reaction specificity and to obtain accurate gene quantification. However, defined target sequences are often not readily available. This situation is particularly evident in the study of rare splice variant transcripts. For gains in efficiency and reaction speed, a small size of PCR amplicon typifies real-time PCR formats, including hydrolysis probes. This study demonstrates the use of oligonucleotides resembling one strand of complete amplicon sequences used in real-time PCR to provide sustainable and precise amounts of the target sequence without the necessity of enlisting nucleic acid cloning procedures. The application of template oligonucleotides is modeled using all of the splice variant forms of human vascular endothelial growth factor.
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ABSTRACT: Canine atopic dermatitis (cAD) is a common condition in dogs that may be a naturally occurring model for human atopic dermatitis (hAD). Despite this, comparative research is limited, particularly into the genetic background of cAD. 1. Measure candidate gene expression in cAD skin using quantitative real time PCR (qPCR). 2. Correlate gene expression to clinical cAD scores (Canine Atopic Dermatitis Extent and Severity Index[CADESI]-03 and intradermal allergen test [IDT]). mRNA was extracted from biopsies of non-lesional and lesional skin from atopic dogs, and healthy skin from non-atopic dogs. Gene expression was quantified using qPCR, and compared between non-lesional atopic, lesional atopic and healthy skin. Gene expression in atopic skin was correlated with clinical severity (CADESI-03) and the number of positive reactions on an IDT. Of the 20 quantified genes, 11 demonstrated statistically significant altered mRNA expression between atopic and healthy skin; dipeptidyl-peptidase-4 (DPP4), phosphatidylinositol-3,4,5-trisphosphate-5-phosphatase-2 (INPPL1), serine protease inhibitor kazal type-5 (SPINK5), sphingosine-1-phosphate lyase-1 (SGPL1), peroxisome proliferator-activated receptor gamma (PPARgamma), S100 calcium-binding protein A8 (S100A8), Plakophilin-2 (PKP2), Periostin (POSTN), Cullin4A, TNF-alpha and metalloproteinase inhibitor-1 (TIMP-1). Three genes correlated with CADESI-03: serum amyloid A 1 (SAA-1), S100A8, and PKP2; and four with IDT results: mast cell protease I (CMA1), SAA-1, S100A8 and SPINK5. Genes with altered expression included those relevant to skin barrier formation and immune function, suggesting both are relevant in the pathogenesis of AD. Many of these genes reflect the proposed pathogenesis in hAD, supporting the use of dogs as a model for hAD. Furthermore, these genes may be considered suitable targets for future genetic and protein function studies in human and canine AD.Journal of dermatological science 05/2009; 55(1):27-33. · 3.71 Impact Factor
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ABSTRACT: Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.Applied and Environmental Microbiology 02/2010; 76(7):2192-202. · 3.95 Impact Factor
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ABSTRACT: Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtDNAs) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtDNA segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtDNAs are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtDNA transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the "mitochondrial bottleneck" in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage.Molecular Biology and Evolution 10/2010; 28(2):949-61. · 10.35 Impact Factor