Storage conditions of intestinal microbiota matter in metagenomic analysis

• Published in: BMC microbiology 2012-07, Vol.12 (1), p.158-158, Article 158
• Main Authors: Cardona, Silvia, Eck, Anat, Cassellas, Montserrat, Gallart, Milagros, Alastrue, Carmen, Dore, Joel, Azpiroz, Fernando, Roca, Joaquim, Guarner, Francisco, Manichanh, Chaysavanh
• Format: Article
• Language: English
• Subjects: Analysis; Biota; Deoxyribonucleic acid; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Electrophoresis, Capillary; Feces - microbiology; Gastrointestinal Tract - microbiology; Generalized linear models; Genes; Genetic aspects; Genomics; Humans; Library collections; Life Sciences; Metagenome - genetics; Metagenomics - methods; Metagenomics - standards; Microbiology; Microbiota (Symbiotic organisms); Molecular weight; Needs for standardization/RNA and DNA degradation/Metagenomics/16S ribosomal RNA; Physiological aspects; Ribonuclease; Ribosomal RNA; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; Specimen Handling - methods; Studies; Taxonomy; Temperature; Time Factors
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/1471-2180-12-158

The structure and function of human gut microbiota is currently inferred from metagenomic and metatranscriptomic analyses. Recovery of intact DNA and RNA is therefore a critical step in these studies. Here, we evaluated how different storage conditions of fecal samples affect the quality of extracted nucleic acids and the stability of their microbial communities. We assessed the quality of genomic DNA and total RNA by microcapillary electrophoresis and analyzed the bacterial community structure by pyrosequencing the 16S rRNA gene. DNA and RNA started to fragment when samples were kept at room temperature for more than 24 h. The use of RNAse inhibitors diminished RNA degradation but this protection was not consistent among individuals. DNA and RNA degradation also occurred when frozen samples were defrosted for a short period (1 h) before nucleic acid extraction. The same conditions that affected DNA and RNA integrity also altered the relative abundance of most taxa in the bacterial community analysis. In this case, intra-individual variability of microbial diversity was larger than inter-individual one. Though this preliminary work explored a very limited number of parameters, the results suggest that storage conditions of fecal samples affect the integrity of DNA and RNA and the composition of their microbial community. For optimal preservation, stool samples should be kept at room temperature and brought at the laboratory within 24 h after collection or be stored immediately at -20°C in a home freezer and transported afterwards in a freezer pack to ensure that they do not defrost at any time. Mixing the samples with RNAse inhibitors outside the laboratory is not recommended since proper homogenization of the stool is difficult to monitor.