Improvement of DNA extraction from human biopsies for a microbiome metagenomic approach

Background: The last few years have seen an accelerated expansion of microbiome studies rendering a clear picture of its major role in human health and disease. Several international studies have shown that the human gut microbiome consists of four enterotypes; however, these studies have not included Latin American population and have not analyzed differences according to diet that can be important microbiome modifiers. On the other hand, few studies have addressed the eye microbiome and microbiome diversity is unknown. In Mexico, Irritable Bowel Syndrome (IBS) and Dry Eye Syndrome are very common disorders and it has been considered that the microbiome is implicated in both of them. Therefore, it is highly important to study the microbiome in these disorders in our population. Methods & Materials: Microbiome composition will be determined by next generation sequencing. Sample standardization is reported herein: DNA extraction was performed from 4 colonic and 1 conjunctiva biopsies, collected at the HGM and the IOCV, respectively. The samples were preserved in RNA later (Ambion) at room temperature and were processed within 3 h of collection. Tissue disruption was conducted with proteinase K enzymatic treatment in buffer AL (QIAamp DNA mini Kit, Qiagen) at 56◦C for 90 min. The extraction was carried out according to the manufacturer’s protocol. The obtained DNA was analyzed with NanoDrop2000 (Thermo Scientific) for DO 260/280nm ratio and for DNA concentration estimation. Also, an agarose gel electrophoresis was run to visualize the DNA integrity. Results: This protocol allowed us to obtain an average DNA quantity of 6 g from the colonic biopsies and 4 g per conjunctivabiopsy with a DO 260/280nm greater than 1.8 and no visible DNA degradation base on literature. Conclusion: The data suggest that this protocol is suitable for obtaining genomic DNA from colonic and conjunctival biopsies for next generation sequencing. This study was supported by research grant IV200315 from PAPIITUNAM.