tissue: rectosigmoid biopsy disease status: HIV-uninfected risk-matched control subject
Treatment protocol
Enrolled subjects were given a mild enema prior to colonoscopy, and rectosigmoid biopsies were obtained between 10 and 20 cm from the anus. Extracted biopsies were snap-frozen in liquid N2 for storage at -80°C. Biopsies were re-suspended in RNAlater ICE (Life Technologies, Carlsbad, CA) according to manufacturer's instructions.
Extracted molecule
genomic DNA
Extraction protocol
Rectosigmoid biopsies were transferred to Lysis Matrix B (MP Biomedicals, OH) tubes containing 600ul RLT buffer (Qiagen, CA). Samples were subjected to 30 seconds of bead beating at 5.5 m/second, followed by centrifugation for one minute at 2000 rpm. Supernatant was transferred to the AllPrep DNA (Qiagen, CA) spin column and nucleic acid purification was carried out according to the manufacturer's instructions. Nucleic acid concentrations were determined using a NanoDrop spectophotometer (Thermo Scientific, MA).
Label
biotin
Label protocol
DNA was resuspended in 96 ul of 30 mM MOPS (morpholinepropanesulfonic acid, pH 7.5), and 4 ul of a 50 mM polyethylene oxide-iodoacetyl biotin (Pierce Chemical) solution was added to introduce the biotin label. The reaction mixture was incubated at 37°C for 1 h, and the labeled DNA was subsequently ethanol precipitated and washed twice with 70% ethanol to remove unreacted biotin compounds.
Hybridization protocol
Hybridization solution contained 100 mM MES (N-morpholinoethanesulfonic acid), 1 M NaCl, 20 mM EDTA, and 0.01% Tween 20, pH 6.6 (referred to as 1X MES). In addition, the solution contained 0.1 mg of herring sperm DNA (Promega)/ml, 0.5 mg of acetylated bovine serum albumin (BSA; Invitrogen)/ml, and 50 pM control oligonucleotide B2 (Affymetrix). Hybridization was carried out at 45°C for 16 h with mixing on a rotary mixer at 60 rpm. Following hybridization, the sample solution was removed and the array was washed and stained in a GeneChip Fluidics Station (Affymetrix). In brief, to enhance the signals, 10 ug of streptavidin (Vector Laboratories)/ml and 2 mg of BSA/ml in 1X MES were used as the first staining solution. After the streptavidin solution was removed, an antibody mix was added as the second stain, containing 0.1 mg of goat immunoglobulin G (Sigma-Aldrich)/ml, 5 ug of biotinylated anti-streptavidin antibody (Vector Laboratories)/ml, and 2 mg of BSA/ml in 1X MES. Nucleic acid was fluorescently labeled by incubation with 10 ug of streptavidin R-phycoerythrin conjugate (Molecular Probes)/ml and 2 mg of BSA/ml in 1X MES.
Scan protocol
The arrays were scanned at 570 nm with a resolution of 3 um with a GeneChip Scanner 3000 (GCS 3000; Affymetrix).
Description
16S ribosomal RNA gene is amplified prior to fragmentation and hybridization.
Data processing
Fluorescence intensities were normalized to spike-in control oligonucleotides provided by SecondGenome, Inc. (San Bruno, CA, USA) using the proprietary PhyCA processing technique, which utilizes aggregate measures of mismatch probe fluorescence intensity as well as distribution of total chip fluorescence (“r scores”). Quartile r score cutoffs were chosen based on r scores retrieved for control probes spiked-in at known concentrations, as recorded from all Phylochip microarrays of the current study, and were as follows: rQ1 > .30, rQ2 > .65, rQ3 > .88.
