Sampling and DNA Isolation in Microbiota Studies

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Microbiota changes, especially intestines in the human body, have been found to be associated with many diseases. One of the most important steps of microbiota studies; transfer, storage and preparation of the specimen under appropriate conditions. Mistakes and omissions that may occur during these stages will seriously affect the result of the study. Many factors such as age, diet, weight, antibiotic use, other medications (metformin, proton pump inhibitors, corticosteroids, etc.), hormonal conditions such as menopause / puberty, smoking / alcohol use, chronic diseases and oral dental problems must be recorded. Much of the work has been done on intestinal microbiota. However, there are also studies focusing on other parts of the body such as mouth, urogenital system, skin, respiratory tract, nose, placenta, breast milk and eye. It is generally preferred to take a fecal sample (at least 1 gram) in intestinal microbiota studies. It can be taken at home or at a health institution. The stool specimen can be stored for up to 24 hours in the room, in the refrigerator (+ 4 ° C) for a few days and at -80 ° C for long periods. If possible, several aliquots should be taken. Quick freezing (at -80°C) after taking sample is reference method. It is best to take it to the stabilization buffer before it is frozen. Rectal and faecal swab specimens have also begun to be used.It is emphasized that there are minor differences according to faecal examples, and it is more convenient and easier for bigger works. However, there is not enough work in this issue yet. Biopsy specimens from colon mucosa and column lavage fl uid may be used, although not very common. Mucosal microbiota is at the surface of the intestinal epithelium. For this reason it is less variant / consistent and interacts directly with host cells. It is therefore emphasized that mucosal biopsy specimens are gold standard sampling types. Nevertheless; colon preparation, invasive procedures such as colonoscopic biopsy, and the effect of biopsy size on the outcome are signifi cant disadvantages of this method. Various samples such as swabs or tissue sumples from other parts of the body can be used in microbiota anealysis. There is no universal DNA extraction method that can fi t all purposes for microbiota studies. High quality pure DNA is required. There are many different commercial kit available. They should be well analyzed before the study and investigated as appropriate. Automated systems have the highest effi ciency. Potential inhibitors that may affect the end must be removed.

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KÖROĞLU, Mehmet. Sampling and DNA Isolation in Microbiota Studies. Journal of Biotechnology and Strategic Health Research, [S.l.], v. 1, p. 50-55, dec. 2017. Available at: <>. Date accessed: 22 feb. 2019.