Oligonucleotides used as template calibrators for general application in quantitative polymerase chain reaction

Department of Immunology, Medical Faculty, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Analytical Biochemistry (Impact Factor: 2.22). 01/2005; 335(2):299-304. DOI: 10.1016/j.ab.2004.09.015
Source: PubMed

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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    • "Primers were synthesised by MWG Biotech and probes were synthesised by Roche Diagnostics using locked nucleic acid analogues with a 5 0 end reporter dye fluorescein (FAM, 6-carboxy fluorescein) and a 3 0 end dark quencher dye. Template oligonucleotides (Mohammadi and Day, 2004) for the amplicon generated by each assay were synthesised by Eurogentec. Two reference (housekeeping) genes (MRPS7 and HIRIP5) were previously identified from micro-array data and confirmed as having stable expression in normal and OA bone and cartilage using a statistical algorithm (Vandesompele et al., 2002). "
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    The Veterinary Journal 11/2007; 179(2):211-8. DOI:10.1016/j.tvjl.2007.08.027 · 1.76 Impact Factor
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    • "Real-time RT-qPCR data was then analysed by using LightCycler® 480 Basic Software (Roche Diagnostics; Lewes, UK). Standard curves were generated for each reference gene by employing cDNA or template oligonucleotides [34], the parameters of which are listed in Table 1. All samples were checked for absence of genomic DNA contamination using a canine genome specific RT-qPCR assay, previously described [25]. "
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    ABSTRACT: Canine atopic dermatitis (cAD) is a common and severe pruritic, inflammatory skin disease that can be considered a naturally-occurring, spontaneous model of human Atopic Dermatitis (hAD). The genetics of cAD are poorly understood and therefore the aim of this project was to investigate the genetic factors involved in the pathogenesis of cAD and to identify specific gene associations with cAD within and between dog breeds. It was hoped that this study would further strengthen the evidence that dogs are a suitable model hAD. Using dogs as a model to study the genetic basis of AD is advantageous because dog breeds form genetically isolated populations exhibiting strong linkage disequilibrium (LD). In contrast to humans where LD across the genome is weak (10-100 kb), domestic dog breeds have strong LD which extends over long distances (0.8 -5 Mb). This is highly advantageous in genetic studies because fewer genetic markers and smaller sample sizes are needed to find disease associations in dogs. To study the genetic basis of cAD a dual approach of candidate gene association study and genome wide association study (GWAS) was used. This gave not only a novel unbiased approach but also used information from previous studies on which gene selection was based. Therefore increasing the likelihood that the causative genes involved in cAD pathogenesis could be identified. This thesis demonstrated altered mRNA expression in 54 genes out of 22,000 transcripts by mRNA microarray in cAD. Further to this qPCR was used to confirm the microarray results and quantify gene expression in potential cAD candidate genes. This approach identified 11 genes with altered expression in cAD. The qPCR results were further correlated 2 with the clinical outcomes: Canine Atopic Dermatitis and Severity Index (CADESI-03) and number of responses on intra-dermal allergen tests. Eleven novel SNPs and 1 novel microsatellite were identified by transgenomic WAVE analysis. The microsatellite was further typed in 659 dogs and but no association with cAD was found. A GWAS with 22,362 SNPs was performed. The significant results were validated by Sequenom along with the SNPs from the candidate gene study (literature selected genes) in 659 dogs across 8 breeds. In total, 232 SNPs across 54 genes and 41 intergenic regions were genotyped on Sequenom. From this 45 putative associations were found in various breeds. A large number of these associations had relevant functions to AD and/or previous association with hAD.
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