Improved procedure for high quality RNA extraction from dust in the indoor environment
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Civil, Environmental and Geodetic Engineering Undergraduate Research Theses; 2019
We spend 90% of our time indoors where we are exposed to microbes and chemicals present in house dust. Some of these harmful compounds include allergens and mycotoxins produced by fungi. These secondary metabolic products are known to have significant, negative effects on human health. However, we do not yet know how changes in environmental conditions can alter gene expression and microbial production of these harmful compounds. Microbial function in dust is difficult to measure because of the high concentration of RNAses, which quickly degrades RNA. RNA extraction is also difficult because the lack of stability in its structure. The purpose of this study was to develop a high quality RNA extraction procedure from house dust for use in the determination of gene expression related to allergen production. As a case study, we considered how different levels of water activity influence gene expression of the common fungal allergen Alt a 1. Water activity levels included 0.5, 0.85, and 1.0 aw, and the indoor setting was simulated by incubating dust samples in carpet pieces in incubators. RNA extraction was performed using Qiagen RNeasy PowerMicrobiome extraction kits. After completing cDNA synthesis and qPCR analysis, we measured expression of allergen Alt a 1 in samples that had been spiked with Alternaria alternata. Modifications to an existing RNA protocol (Qiagen) intended for fecal samples improved the RINe score from below detection to 7.5. We confirmed production of Alt a 1 in all three samples incubated at 1.0 aw, 1 of the 3 samples incubated at 0.85 aw, and none of the samples incubated at 0.50 aw. Overall, this work overcame obstacles related to high-quality extraction of RNA from house dust. Future work will demonstrate important implications for moisture control in indoor environments in regards to allergen production and chronic exposure.
Academic Major: Food, Agricultural, and Biological Engineering
Alfred P. Sloan Foundation
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