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Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

ABSTRACT: BACKGROUND:Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles. METHODS:Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing. RESULTS:The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles. CONCLUSION:Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

SUBMITTER: Cuthbertson L

PROVIDER: S-EPMC4793934 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

Cuthbertson Leah L Rogers Geraint B GB Walker Alan W AW Oliver Anna A Hoffman Lucas R LR Carroll Mary P MP Parkhill Julian J Bruce Kenneth D KD van der Gast Christopher J CJ

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 20141024 4

<h4>Background</h4>Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.<h4>Methods</h4>Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze- ...[more]

PMID: 25459563

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Project description:Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.

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Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

Publications

Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

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