Rheumatoid Arthritis Research Today is a free monthly online journal that collates and summarizes the latest research about Rheumatoid Arthritis, including details on treatment, symptoms, causes, medication. | ||||||||
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A rapid semi automated method for DNA extraction from dried-blood spots: application to the HLA-DR shared epitope analysis in rheumatoid arthritis.Pachot A, Barbalat V, Marotte H, Diasparra J, Gouraud A, Mougin B, Miossec P Hospices Civils de Lyon, BioMérieux Joint Unit, Hôpital Edouard Herriot, 5, Place d'Arsonval, 69003, Lyon, France. Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, "DBS-miniMAG", that combines filter paper dried blood spots (DBS) with the NucliSens miniMAG semi-automated instrument (bioMérieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in a cohort of 72 arthritis patients. This new method was compared to the standard manual DBS extraction protocol using FTA reagents (Whatmann Bio-Science), and to a reference phenol-chloroform-based method using EDTA whole blood samples. Higher yield of PCR amplicons was observed with DNA extracts obtained using "DBS-miniMAG" method. The intra- and inter-assay variability of the "DBS-miniMAG" method was similar to that obtained with "DBS-FTA" washing process. Concerning the HLA-DR SE genotyping, "DBS-miniMAG" and "DBS-FTA" methods gave 100% concordance compared to the reference phenol-chloroform method. More importantly, the hands-on time and the turnaround time for "DBS-miniMAG" were both two-times shorter than for "DBS-FTA" protocol. Therefore, the "DBS-miniMAG" combination could facilitate polymorphism analysis in routine clinical practice and the creation of large DNA banks using very small amounts of blood. Published 5 November 2007 in J Immunol Methods, 328(1): 220-5.
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