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DNA extraction by FTA™ technology: application for rapid detection of 1p/19q deletions in gliomas

Allelic losses on chromosomes 1p and 19q are the most characteristic genetic alterations found in oligodendrogliomas [1–3]. Their association with a higher chemosensitivity remains uncertain [4–6] but these deletions seem to be associated with a better survival in patients [4,5]. Different techniques are available for detection of 1p/19q deletions, the most often used being loss of heterozygosity analysis (LOH), fluorescent in situ hybridization and comparative genomic hybridization analysis [1,3]. Nevertheless, 1p/19q analysis is not carried out routinely in most medical centres. We have adapted FTATM technology, commercially available reagent-loaded papers similar to filling cards, to simplify DNA extraction from both leucocytes and tumours before LOH analysis. This strategy involves four successive steps: (i) samples are applied to the FTATM card, cells lysed and DNA becomes entangled in the paper; (ii) a small disc is removed from the card and washed to remove proteins; (iii) the disc is placed directly in the polymerase chain reaction (PCR) mix and PCR amplified in situ; (iv) microsatellite analysis is performed on an automated sequencer (Figure 1). Tumour samples and blood were collected from a consecutive prospective series of 70 patients with a glioma, undergoing a resection (41 cases) or a stereotactic biopsy (29 cases), before any other treatment. All patients had given informed consent. Diagnosis was established according to World Health Organization classification [2]. Fresh or frozen blood after thawing (75 ml), collected with EDTA, was loaded onto the FTATM card (Transgenomic, Crewe, UK). Fresh (60 cases) or frozen tumour tissues (10 cases) after thawing at room temperature (2 mm) were crushed with parafilm onto the card. A critical step is to ensure that samples applied are dry before taking a punch. A fragment of each sample applied on the card was smeared onto glass slides, fixed in 100% ethanol and stained with haematoxylin-eosin in order to verify that DNA was extracted from an area with a high density of tumour cells. After drying at room temperature, sample discs (1.2 mm diameter) were removed from the FTATM card, transferred to PCR tubes, and 150 ml of FTA Purification Reagent (FPR) was added, according to the manufacturer’s instructions. After incubation (5 min, room temperature), FPR was removed using a pipette. This step was repeated twice. Then, 150 ml of sterile water was added on FTATM disc, incubated for 5 min, and then removed using a pipette. This step was repeated twice. The disc was dried for 1 h at room temperature or for 10 min at 56°C, before performing PCR amplification. For microsatellite analysis, we used the 1p D1S508 and D1S2734, and the 19q D19S219 and D19S412 chromosomal markers, whose primer sequences have been previously reported [6,7]. For each microsatellite, primer mixes contained 200 ng of a reverse primer and 200 ng of a forward primer mix (9/10 of unlabelled primer and 1/10 of the same primer labelled with the fluorescein dye 6-carboxyfluorescein). PCR was performed in a final volume of 50 ml containing 0.25 unit of Hot Star Taq DNA polymerase (Quiagen, Courtaboeuf, France). The D19S219 and D19S412 markers were amplified after a 15-min denaturation at 95°C, 30 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, and a final 5 min extension at 72°C. The D1S508 and D1S2734 markers were amplified in the same conditions, except for annealing temperatures (48°C and 50°C respectively), no extension step and 35 cycles. D19S219 and D19S412 reaction products (1 ml each) were pooled in a mix containing 20 ml of deionized formamide and 0.1 ml of 400HD ROX (PE Applied Biosystems, Perkin Elmer, Foster City, CA, USA). D1S508 and D1S2734 reaction products were pooled in the same conditions. After denaturation for 10 min at 94°C, samples were analysed on an Applied Biosystems 310 automated sequencer. When microsatellites were non-informative because of constitutional homozygosity, 1p (D1S468, D1S513) and 19q (D19S596 and D19S112) additional markers were used. We analysed a series of 70 gliomas, including 44 astrocytomas (four grade II, four grade III, 36 glioblastomas), 20 oligodendrogliomas (seven grade II, 13 grade III) and