Total RNA was prepared from the P. aeruginosa strains using the same as procedure as described for qRT-PCR, except that a greater volume of culture was used. The quality of the purified RNA was confirmed using an Agilent 2100 Bioanalyzer System. cDNA was generated and labeled using the Bioprime labeling kit (Invitrogen), and the microarray hybridization was performed using Hybridization solution (MYcroarray.com). The microarray data were normalized and analyzed using Genowiz 4.0 (Ocimum Biosolutions, India). Total 5544 genes were analyzed, and among these, the comparative transcriptional profile of HAQ-related genes between WT and desB mutant was used for data interpretation.
Bioprime labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BioPrime labeling kit is a laboratory product designed for the labeling of biomolecules, such as DNA, RNA, and proteins, for a variety of analytical and research applications. The kit provides the necessary reagents and materials to perform this labeling process efficiently and effectively.
Automatically generated - may contain errors
9 protocols using bioprime labeling kit
1
Transcriptomic Analysis of P. aeruginosa
2
Comprehensive Genomic Profiling by CGH
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DNAs were digested for 30 minutes with DNase I prior to Klenow-based labeling. In each case, 1 μL of 10× DNase I reaction buffer and 2 μL of DNase I dilution buffer were added to 7 μL of DNA sample and incubated at room temperature and then transferred to 70°C for 30 minutes to deactivate DNase I. Sample and reference templates were then labeled with Cy-3 dUTP and Cy-5 dUTP, respectively, using a BioPrime labeling kit (Invitrogen) according to our published protocol (56 (link)). All labeling reactions were assessed using a Nanodrop assay (Nanodrop) prior to mixing and hybridization to CGH arrays (Agilent Technologies) for 40 hours in a rotating 65°C oven. All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings. The CGH data were assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2) (57 (link)).
3
Visualization of Xist and CIZ1 in Cells
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Female cultured cells were processed for the detection of Xist transcript (red) by RNA-FISH followed by immuno-FISH for CIZ1 (green) using N-terminal antibody 1794. An 11-kb Spe1–Sal1 mouse Xist fragment was fluorescently tagged with Cy3-dUTP (GE Healthcare) using BioPrime labeling kit (Invitrogen). Samples were incubated with probe overnight at 37°C. For subsequent detection of CIZ1, antibody 1794 was applied for 1 h followed by secondary anti-rabbit FITC (Sigma) for 1 h. Cells were imaged and processed using Adobe Photoshop CS4 to enhance signal definition. Prior RNA-FISH processing resulted in reduced CIZ1 signal intensity throughout the nucleus. For SR 3D-SIM and RNA-FISH on splenocyte Xist, cDNA was labeled with Spectrum green-dUTP or Spectrum red-dUTP by nick translation (Abbott Molecular). Following fixation and permeabilization, cells were incubated with primary antibody for 1 h and then with Alexa fluor goat anti-rabbit 594 for 30 min and then washed and post-fixed before detection of Xist overnight. After extensive washing, the cells were incubated with 2 µg/mL DAPI and mounted with VectorShield.
4
aCGH Analysis of Genomic DNA
5
Genomic DNA Profiling via aCGH
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Patient samples were flow sorted prior to genomic analysis using our published DNA content based protocols [21 (link)]. Briefly, DNAs were extracted using QIAGEN micro kits (Valencia, CA, USA). For each hybridization, 100 ng of genomic DNA from each sample and of pooled commercial reference (Promega, Madison, WI, USA) were amplified using the GenomiPhi amplification kit (GE Healthcare, Piscataway, NJ, USA). Subsequently, 1 ug of amplified sample and 1 ug of amplified reference template were digested with DNasel then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA, USA). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE, USA) prior to mixing and hybridization to CGH arrays with either 244,000 or 400,000 oligonucleotide features (Agilent Technologies, Santa Clara, CA, USA). The aCGH data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (accession numbers GSE54328 and GSE21660).
6
Comparative Genomic Hybridization Protocol
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DNA was DNAse I digested, labeled using a BioPrime Labeling Kit (Invitrogen) using Cy-5 dUTP for the sample and Cy-3 dUTP for the reference genome, hybridized to 400k comparative genomic hybridization (CGH) arrays (Agilent Technologies), scanned using an Agilent 2565C DNA scanner, and the images were analyzed with Agilent Feature Extraction v11.0 using default settings.
7
Genome-Wide DNA Copy Number Profiling
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DNAs were extracted using QIAGEN micro kits (Valencia, CA, USA). For each hybridization, 100 ng of genomic DNA from each sample and of pooled commercial 46XX reference (Promega, Madison, WI, USA were amplified using the GenomiPhi amplification kit (GE Healthcare, Piscataway, NJ, USA). Subsequently, 1 μg of amplified sample and 1 μg of amplified reference template were digested with DNaseI then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA, USA). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE, USA) prior to mixing and hybridization to CGH arrays with either 244,000 or 400,000 oligonucleotide features (Agilent Technologies, Santa Clara, CA, USA). The aCGH data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (accession numbers GSE54328 and GSE21660).
8
Optimized aCGH Protocol for Frozen and FFPE Samples
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All aCGH was done according to our published protocols21 (link)–24 (link). Briefly, DNAs from frozen tissue and FFPE samples were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight templates were digested for 30 min while DNAs from FFPE samples were digested for 1 min. In each case 1 µl of 10 × DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70 °C for 30 min to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols23 (link). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400 k CGH arrays (Agilent Technologies, Santa Clara, CA) for 40 h in a rotating 65 °C oven. After washing microarrays were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2)25 (link).
9
Comparative Genomic Hybridization Protocol
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DNA was extracted using Qiagen (Hilden, Germany). For each hybridization, 100 ng of genomic DNA from each sample and pooled commercial 46XX (Promega) were amplified using the GenomiPhi amplification kit (GE Healthcare). Subsequently, 1 mg of amplified sample and 1 mg of amplified reference template were digested with DNaseI and labeled with Cy-5 deoxyuridine triphosphate and Cy-3 deoxyuridine triphosphate, respectively, using a BioPrime labeling kit (Invitrogen). All labeling reactions were assessed using a Nanodrop assay before mixing and hybridization to either the 244,000 or 400,000 feature CGH arrays (Agilent Technologies). The array comparative genomic hybridization (aCGH) data were assessed using a series of quality control metrics and analyzed using an aberration detection algorithm. 27 The latter identifies all aberrant intervals in a given sample with consistently high-or low-log ratios according to the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log 2 ratios from its expected value of 0 and is proportional to the height (absolute average log ratio) of the genomic interval and the square root of the number of probes in the interval.
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