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Paraffin blocks were sectioned onto SuperFrost Plus slides, dried overnight, and baked for 1 h at 60 C before immediately proceeding with the RNAScope Multiplex Fluorescent v2 assay according to the manufacturers instructions with the following modifications: Target retrieval was performed for 15 min in a boiling beaker, Protease III was used for 30 min pre-treatment at 40 C and hybridized slides were left overnight in 5 SSC before proceeding with the amplification and labeling steps

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Because the tobacco culture, including tobacco production and use, runs deep in Central Appalachia, it is not surprising that tobacco use disproportionately impacts rural adolescents from this region 14,38,39 , contributing to one of the worst health profiles in the US 24,26-33