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


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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