Dataset Information

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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Publications

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.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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