Although the subjects could be asked to mix more thoroughly their stool after collection, this

requirement is difficult to monitor. Therefore, the use of RNAse inhibitors may not be the best choice for semi or large-scale studies. Conclusions Our study, although under a context of a small sampling size and other limiting parameters, suggests that storage conditions of stool samples can largely Trichostatin A affect the integrity of extracted DNA and RNA and the composition of their microbial community. In light of our observations, our recommendation for semi or large-scale metagenomic and metatranscriptomic projects is to keep the samples at room temperature and to bring them in the laboratory within the initial 24 Ku-0059436 molecular weight hours after collection. Selleckchem Fedratinib Alternatively, if bringing the samples during this period is not possible, samples should be stored immediately at −20°C in a home freezer. In this case, samples need to be transported afterwards in freezer packs to ensure that they do not defrost at any time.

Mixing the samples with RNAse inhibitors and keeping them at home for longer periods of time (days) is not recommended since proper homogenization of the stool is difficult to monitor outside the laboratory. Methods Samples Fecal samples were collected from healthy volunteers (n = 11), who did not receive antibiotics within the last three months. Samples were stored following 3 different procedures, which took into account volunteer’s compliance. In the first procedure, before being frozen at −80°C, each sample was kept at room temperature (RT) during different time periods (3 h, 24 h, 48 h, 72 h and 14 days). Time points before 3 h were not applicable, since volunteers needed this time to bring the samples from home to the laboratory. In the second protocol, samples were immediately frozen by the volunteers at their home freezer at −20°C and later were brought at the laboratory in a freezer pack, where they were immediately stored

at −80°C. In order to test the effect of freezing and thawing episodes, some aliquots were defrosted during 1 h and 3 h before being stored at −80°C. In the third protocol, some volunteers agreed to collect their samples in tubes containing the RNAse inhibitor RNA isometheptene Later® (Ambion) as indicated by the manufacturer instructions. The tubes were kept at room temperature during different time periods (3 h, 24 h, 14 days and 1 month) before RNA extraction. The protocol was approved by the Ethics Committee of the Vall d´Hebron University Hospital and all participants gave informed consent. Assessing the quantity and quality of total RNA For total RNA extraction, we modified the protocol described in Zoetendal et al. [15], which utilizes 15 g of fecal sample. Briefly, 200 mg of fecal sample were mixed with 500 μl TE buffer, 0.8 g Zirconia/silica Beads, 50 μl SDS 10% solution, 50 μl sodium acetate and 500 μl acid phenol.