RNA-seq in Pulmonary Medicine: How Much Is Enough?

Abstract

RNA-seq is eclipsing microarray techniques to measure gene expression in tissues (1) because of several factors including reduced costs, increased sensitivity (1, 2), and the ability to detect splice variants (1) and nonhuman sequences in the sample (3). We investigated the use of RNA-seq to assess the transcriptome of three types of tissues relevant to pulmonary research: bronchial brushes obtained from healthy volunteers, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface, and healthy whole-lung samples obtained from the Lung Tissue Research Consortium (LTRC). Our analysis suggests that 40-60 million reads are sufficient to assess the majority of the host transcriptome in these samples, whereas higher depths are needed for very low expressed genes or rare splice variants. We also analyzed read depths of human bronchial epithelial (HBE) cells treated with IL-13 or IL-17 to determine whether inflammation affects the parameters. Treatment with IL-13 or IL-17 showed no significant effect on the results, suggesting that 40 million reads is adequate to assess the transcriptome of HBE cells in either resting or cytokine-stimulated conditions. Methods RNA sample collection and isolation. Healthy control participants were recruited as part of the National Heart Lung and Blood Institute's Severe Asthma Research Program (4). Healthy controls were defined as healthy atopic or nonatopic individuals with normal lung function and without a history of chronic respiratory conditions, including rhinitis requiring topical corticosteroid treatment. A demographics table is included as Table E1 in the online supplement. Bronchial brushings were obtained from the fourth-to sixthgeneration airways, as previously described (5). Bronchial brushings were placed directly into ice-cold Qiazol (Qiagen, Valencia, CA) during the bronchoscopy to ensure minimal RNA degradation, after which samples were stored at 280 8 C until RNA isolation, using Qiagen RNeasy isolation kit (Qiagen). RNA was quantitated using nanodrop, and integrity was determined with a total RNA nano chip (Agilent Technologies). The National Heart, Lung, and Blood Institute LTRC obtained lung biopsies from patients with chronic obstructive pulmonary disease undergoing lung volume reduction surgery, transplantation, or resection for malignancies. In the latter case, tissue from nontumorous portions were collected. Patient phenotyping was determined by various methods including medical questionnaires, chest computed tomography scans, spirometry, 6-minute-walk tests, cardiopulmonary exercise tests,

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