Nanodrop nd 1000

All the oligonucleotide probes were synthesized by Thermo Fisher Scientific (Ulm, Germany). 16 S rRNA gene was amplified by using a Biometra Thermal Cycler II (Biometra, Gottingen, Germany) and 2.5 U of GoTaq Flexi polymerase (Promega, Madison, WI) in a final volume of 50 μL following the protocol described in Cruciani et al.^{26 (link)}. PCR products were purified by using a High Pure PCR Clean up Micro kit (Roche, Mannheim, Germany), following the manufacturer instructions, and quantified by NanoDrop ND-1000.

DNA was extracted from 1 mL of peripheral blood samples using a DNA automatic extractor (MagNa Pure^® System, Roche Applied Science, Indianapolis, IN, USA) and quantified spectrophotometrically in NanoDrop^® ND-1000 (Wilmington, Delaware). The 260/280 absorbance ratio was used to measure the purity of the samples.
The genotyping of ABCG2 (rs2231142), SLC2A2 (rs8192675), SLC22A2 (rs316019), SLC22A3 (rs3088442), SLC22A4 (rs272893, rs1050152), SLC29A4 (rs3889348), SLC47A1 (rs2289669), and SLC47A2 (rs12943590) was outsourced and performed by MALDI-TOF mass spectrometry, with the MassARRAY^® platform (Agena Bioscience Inc., San Diego, CA, USA), at CEGEN-PRB3-ISCIII. All assays were performed with internal quality control, with a genotyping success rate and reproducibility of 100%. The genotyping of ABCB1 (rs1128503, rs1045642 and rs2032582) and SLC22A1 (rs72552763, rs12208357, and rs34059508) was performed at the Research Unit of Hospital Universitario de Burgos using TaqMan^® probes in a ViiA7 qPCR instrument (ThermoFisher, Waltham, MA, USA). The genes and polymorphisms were chosen for their implication in metformin transport and the functional consequences of the polymorphisms based on previous literature and minor allele frequencies.