Genomic dna enzymatic labeling kit

Array-based CGH was performed by the manufacturer Takara Bio Dragon Genomics Center (Seta, Shiga, Japan). In brief, DNA (2 micro g) was fluorescent-labeled by random priming DNA synthesis in the presence of Cy3-dUTP (control group) or Cy5-dUTP (patient group) (Genomic DNA Enzymatic Labeling Kit; Agilent Technologies, Hachioji, Tokyo, Japan). DNA labeling efficiency was estimated by spectrophotometry (Nanodrop ND-2000®) measuring optical absorbance at 260 nm for DNA, at 550 nm for Cy5, and at 649 nm for Cy3. Cy5- and Cy3-labeled DNAs were randomly paired, mixed, and hybridized to SurePrint G3 Human CGH Microarrays (1 M) in the presence of human Cot-1 DNA (Oligo aCGH/ChIP-on-chip Hybridization Kit, Agilent Technologies). Following hybridization for 24 h, microarray slides were washed according to the manufacturer’s instructions and immediately scanned on a DNA Microarray Scanner (Agilent Technologies). With the given limitation of the sample number, we took an advantage of the above direct comparison between case and control samples [57 (link)]. This approach allowed us to determine relative ratios of their gene dosages but not their absolute gene dosages. However this procedure decreased data deviations, compared with the CGH analysis utilizing two microarrays and reference genome DNA [30 (link)].

Negative screening was performed using the Decode RNAi Pooled Lentiviral shRNA Screening Libraries: Annotated Genome Negative Selection Kit (Thermo Scientific). Briefly, T-REx HeLa cells (Invitrogen) were infected with a lentiviral siRNA expression library (Thermo Scientific) using TransDux reagent (System Biosciences). Green fluorescent protein-positive cells were selected by puromycin treatment and divided into two populations, one of which was irradiated with 0.75 Gy using a ¹³⁷Cs irradiator (Gy/min, Best Theratronics); the other served as a non-irradiated control. Four days after irradiation, genomic DNA was purified from each population using a DNA purification kit (Dojindo). Barcode sequences were amplified from 0.5 μg of genomic DNA, purified by gel extraction, and labeled using a Genomic DNA Enzymatic Labeling Kit (Agilent Technologies). After purification using Amicon Ultra-0.5 ml centrifugal filters (Millipore), the labeled barcode sequences were hybridized with microarray slides for 17 h and the slides were then washed according to the Agilent CpG microarray protocol. Cluster analysis of the extracted genes was performed using the information in the GeneCards database (http://www.genecards.org/) and KEGG (http://www.genome.jp/kegg/).

Amplification and labeling of RNA from single fibers were fully described in our previous work [27 (link)]. Briefly, RNA was purified from each single myofiber and exponentially amplified using the TransPlex Whole Transcriptome Amplification 2 Kit (Sigma-Aldrich). RNA was reverse-transcribed in a cDNA library, and the library was amplified for 18 cycles. cDNA was then labeled by using the Genomic DNA Enzymatic Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Labeled cDNA was purified using the Amicon 30 kDa filters (Millipore, Burlington, MA) and quantified. Microarray experiments were performed as previously described [27 (link)] with SurePrint G3 Mouse Gene Expression 8 x 60 K microarray platforms (Agilent Technologies).

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.

CGH array was performed using SurePrint G3 Human CGH Bundle (4 × 180K) (Agilent) according to manufacturer instructions. Briefly, 1 μg of genomic DNA corresponding to either a human male control [20] , EndoC-βH2 or EndoC-βH3 cells both at passage 40 was fragmented by heating at 95 °C for 30 min. Fragmented DNAs were labeled with Cy3 (control DNA) and Cy5 (EndoC-βH2/EndoC-βH3 DNA) fluorescent dUTP, respectively, using Genomic DNA Enzymatic Labeling Kit (Agilent Technology). Microcon YM 30 spin columns (Millipore) were used to remove the unincorporated nucleotides and dyes. Hybridizations of labeled DNA to SurePrint G3 Human CGH Bundle (4 × 180K) array (Agilent) were performed in a hybridization oven at 42 C at 20 rpm for 40 h. Hybridized arrays were then washed following the manufacturer's instructions. Microarray slides were scanned on a Nimblegen MS200 Microarray Scanner at a 2 μm resolution. Feature extraction was done with Cytogenomics Software (Agilent). Extracted data were imported and analyzed using Nexus 7.0 (Biodiscovery).