**2. The importance of sampling**

Microbiota composition is typically studied in biological samples, such as stool, mucosal biopsy, intestinal aspirate, luminal brushing, etc.; for population studies, however, sampling should be noninvasive and performed mainly in healthy conditions. In practice, although fecal samples are the most used as a representation of the intestinal tract, significant differences have been demonstrated among the mucosal microbiota of each intestinal anatomic region [10, 11]. Its main limitations are that feces also contain DNA from microorganisms ingested with food, which are not part of our microbiota; that we are not able to differentiate between living and dead microorganisms; and given that diet causes fluctuations in its composition, longitudinal sampling is required to decipher the real core of the native microbiota.

After deposition, strict anaerobic bacteria begin to lose viability after contact with oxygen, an irrelevant factor if DNA techniques are used. However, numerous studies have demonstrated the influence on the results of collection, transport, storage, and processing of the samples [12]. All samples belonging to the same study should be collected, preserved, and processed simultaneously and identically, to minimize any source of variability [13]. As a rule, recommendations on feces collection consist of using a sterile container with a screw cap, which should be transported without delay to the processing center and frozen as soon as possible at −80°C, although it is also acceptable to perform a first freeze at higher temperatures (as low as −20°C). Freezing prevents changes in microbial communities until nucleic acid extraction can be performed, which is crucial for RNA analysis because it is more easily degraded

than DNA in freeze–thaw cycles. Therefore, optimizing sampling methods should not be ignored. Future sampling procedures should include reducing invasiveness, performing non-cross-contamination sampling, and minimizing disturbance to normal intestinal physiology [12].
