Hi rpm buffer

The procedures for DNA digestion, labeling, and hybridization for the oligo arrays were performed according to the standard Agilent protocol v7.1, with minor modifications. Briefly, 0.25 µg of patient genomic DNA and 0.25 µg of pooled gender-matched reference DNA (Promega) were labeled with Cyanine 5 (Cy5) or Cyanine 3 (Cy3) dyes (Agilent). Following purification with Amicon 30 kDa filters (Millipore), the labeled DNA yield and dye incorporation were measured using an ND-2000 spectrophotometer (NanoDrop). The Cy3-and Cy5-labeled samples along with 2 µg human Cot-1 DNA (Invitrogen), 10X blocking agent (Agilent) and 2X Hi-RPM Buffer (Agilent) were added and hybridized together at 65°C on the CancerArray (Ambry Genetics) for 24 h in a rotisserie oven at 20 rpm. Slides were washed according to the manufacture's protocol and scanned at 3 µm resolution on an Agilent G2565CA high-resolution DNA microarray scanner.

cDNA preparations that were positive for S. suis orf2 by qPCR were used for microarray experiments. cDNA (0.5–2.5 µg) was labeled with cyanine 3-d-UTP using the Genomic DNA Enzymatic Labeling kit (Agilent Technologies) according to manufacturer’s instructions and purified using Amicon Ultra-30K membrane filters (Millipore). Prior to hybridization, pure labeled cDNA in Agilent blocking reagent and Agilent Hi-RPM buffer was incubated for 3 min at 95°C followed by 30 min at 37°C. The mixture was then hybridized to one array of an 8 × 15 k custom S. suis oligo-array (Agilent Technologies) containing probes based on genome sequences of strain P1/7 [27] and probes designed on incomplete genome sequences of strains 891,591, 8067, 7917, and 6388 and incubated at 65°C in a rotator hybridization oven. After 17 h, arrays were washed for 5 min at RT in Oligo aCGH wash buffer 1 (Agilent Technologies) and subsequently for 1 min at 37°C in Oligo aCGH wash buffer 2. Arrays were dried at RT and scanned using a Surescan high-resolution DNA microarray scanner (Agilent Technologies), followed by data analysis using Feature extraction software and Genespring GX software, both also from Agilent Technologies.