Alconox detergent

Screening of known North African mutations associated with USH was performed with a cost-effective NADf chip using multiplex-PCR coupled with dual-color arrayed primer extension as described by Chakchouk et al. [16 (link)]. Multiplex-PCR was performed in a total volume of 50 μl, including 40 μl of the 12 multiplex PCR fragmented products, 0.5 μl of each fluorescent ddNTP, and 1 U of thermosequenase (GE Healthcare, Chalfont St. Giles, UK). Arrays were hybridized with the reaction mixture for 30 min at 60 °C using the FAST Frame Cassette (Sigma-Aldrich, Dorset, UK). The arrays were then washed briefly with 0.3% Alconox detergent (Sigma-Aldrich, Munich, Germany) and distilled water. Slides were then dried before scanning. The coding exons and flanking intronic sequences of all three USH1 genes (MYO7A, USH1C, and PCDH15) were amplified using forward and reverse primers. Mutations were identified by sequencing the PCR products from one affected individual from each family on an ABI Prism 3100-Avant automated DNA Analyzer (Applied Biosystems). Exons harboring mutations were amplified on 40 ng genomic DNA, and then either Sanger sequencing or PCR-restriction fragment length polymorphism (RFLP) analysis was used to examine whether the mutations segregated with the disease in the families and were not present in the 50 control individuals.

A meltblown PEF was purchased from Gwangil Textile (Daegu, Korea). Arizona fine test dusts (ISO 12103-1 A2 fine grades) were acquired from Powder Technology Incorporated (Minnesota, USA). The concentrations of fine dusts were ∼80 μg m⁻³ for PM_1.0 (PM diameters below 1.0 μm), ∼500 μg m⁻³ for PM_2.5–1.0 (PM diameters between 1.0 and 2.5 μm), and ∼1000 μg m⁻³ for PM_10–2.5 (PM diameters between 2.5 and 10 μm). Ethanol (99.5%) was obtained from Samchun Chemicals (Seoul, Korea). The alconox detergent was purchased from Sigma-Aldrich (Missouri, United States). Deionized (DI) water (18.3 MΩ cm⁻¹) was obtained from a reverse osmosis water system (Human Science, Korea). Teflon sheets (10 cm × 10 cm) and cylindrical blocks (diameter: 4.3 cm, height: 5.6 cm, mass: 57 g) were acquired from Yangzhong Haiteng Fluorine Plastic Product Factory (Shanghai, China) and Vitlab (Grossostheim, Germany), respectively.