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The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data.

ABSTRACT: Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

SUBMITTER: Korenkova V

PROVIDER: S-EPMC4935944 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods.ResultsFourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods.ConclusionOur results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.

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The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data.

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