Human genechip 1.0 st array

RNA was extracted from GI biopsies by the RNeasy Plus Kit (Qiagen, Germantown, MD). Expression of B12 pathway-encoding genes was assessed with the Human GeneChip 1.0 ST Array (Affymetrix, Santa Clara, CA). Only samples with adequate RNA quantity and quality (>100 ng/μl and RNA integrity number ≥7, as suggested by the manufacturer) for both preoperative and postoperative biopsies were included in microarray analysis.
Genes of interest with altered expression levels by microarray analysis were validated by RT–qPCR (adequate RNA samples defined as those with RNA integrity number ≥5).^{12 (link)} Candidates for gene validation were selected based on originality: either the change in expression after RYGB was not reported previously or the gene was expressed at a different site than is normally observed. Genes expressed at multiple sites were validated at the most physiologically relevant site. RT–qPCR was developed with TaqMan gene expression assays (Thermo Fisher Scientific, Waltam, MA), using beta-actin as a reference gene. From the 20 patients selected, RT–qPCR was performed in a larger sample size than microarray technique, as described in black blocks in Figure 1. In addition to the 20 patients selected in the study, 5 other patients were selected for gene expression validation by RT–qPCR.