Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification |
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Authors: | Josep L Acero Sanchez Olivier Y F Henry Teresa Mairal Nadja Laddach Anders Nygren Siegfried Hauch Jasmin Fetisch Ciara K O’Sullivan |
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Institution: | 1. Nanobiotechnology and Bioanalysis Group, Departament d’Enginyería Quimica, Universitat Rovira i Virgili, 26 Avinguda Pa?sos Catalans, 43007, Tarragona, Spain 2. MRC Holland, Willem Schoutenstraat 6, 1057 DN, Amsterdam, The Netherlands 3. AdnaGen AG, Ostpassage 7, 30853, Langenhagen, Germany 4. Institució Catalana de Recerca i Estudis Avan?ats, Passeig Lluís Companys, 08010, Barcelona, Spain
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Abstract: | An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer,
extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented.
In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified
by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the
surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe
sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step
was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed
by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and
limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L−1, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific
targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique
typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out,
relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it
very promising for the analysis of MLPA products. |
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