Use of Rhino-Probe™ sampling for analyzing cross-shift variation in the expression of IL8, IL6 and TNF alpha in nasal epithelial cells from Colorado dairy workers

Margaret Davidson, Brie Hawley, Joshua Schaeffer, Mary Bradford, John Volckens, Stephen J. Reynolds

Research output: Chapter in Book / Conference PaperConference Paper

Abstract

![CDATA[Introduction: Dairy work with its inherent exposure to bioaerosols, has long been associated with adverse inflammatory conditions in workers. In previous studies, dairy worker response to pro-inflammatory bioaerosols has been assessed by analysis of proteins (MPO, TNFα and IL8 etc) in nasal lavage fluid. Recent clinical studies have used Rhino-Probe Nasal Curettes to study inflammatory responses from exposure tobacco smoke and diesel exhaust. The object of this study was to utilize Rhino-probes™ in conjunction with RT-PCR to study the inflammatory response of Colorado dairy workers exposed to bioaerosols. Methods: Samples of nasal epithelial cells were collected from16 dairy workers pre and post workshift. Samples were stored in RNAlater (Qiagen) at -74°C. RNAlater was removed by adding phosphate buffer and pelletizing the cells by centrifuging. The supernatant was transferred to RNA lysis (RLT) buffer (Qiagen, RNeasy Micro Kit) and extracted with Qiagen QIAshredder and RNeasy Micro Kits. Total RNA content was quantified by Nano-Drop. mRNA transcripts for candidate genes GAPDH (housekeeping), IL8, IL6 and TNFalpha mRNA transcripts were quantified by RT-PCR (Bio-Rad iScript™ One-Step RT-PCR Kit with SYBR® Green; Instrument Bio-Rad CFX model). Results: A total of 32 paired (Pre and Post) samples were collected from the volunteers, 15 Hispanic males and 1 female. Of these, on 12 paired samples underwent RT-PCR. The remainder were rejected due to poor RNA yield and/or quality scores (260:280) of <1.7. A mean (standard error) cross-shift increase was observed for IL8 mRNA transcripts 1.78 ct (0.66 n=8) and 0.65 (0.77 n=7) for TNF alpha. IL6 was below the limit of detection in all but one sample. Conclusions: The mean cross-shift increase in IL8 and TNFa transcripts in dairy workers exposed to bioaerosols is consistent with the literature from previous studies measuring proinflammatory cytokines in nasal lavage and venous blood of dairy farmers. Over two thirds of the samples collected could not be analyzed due to poor RNA yields and quality. The quantity of tissue collected was limited because workers were uncomfortable with the probes and the accumulation of dust and mucous made it difficult to access the nose. In addition, the complete removal of RNAlater from tissue samples was problematic due to its viscosity, and traces were carried over to the RNA elution column, which may have affected the quality of RNA extracted. A less confronting sampling technique is recommended, and RNAlater be avoided for Rhino-Probe samples. Alternatively, samples could be transferred to RLT buffer and snap frozen in the field with dry ice. This will reduce the sample extraction time where RNA degradation may occur and eliminate potential downstream interference.]]
Original languageEnglish
Title of host publicationNordic Meeting on Agricultural Occupational Health and Safety (NMAOHS), 25-27 August 2014, Porvoo, Finland
PublisherFinnish Institute of Occupational Health
Pages20-20
Number of pages13
Publication statusPublished - 2014
EventNordic Meeting on Agricultural Occupational Health and Safety -
Duration: 1 Jan 2014 → …

Conference

ConferenceNordic Meeting on Agricultural Occupational Health and Safety
Period1/01/14 → …

Keywords

  • airborne pollutants
  • dairy workers

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