Cy3 ctp

Manufactured by PerkinElmer

Sourced in United States

Cy3-CTP is a fluorescently labeled nucleotide used in various molecular biology applications. It can be incorporated into nucleic acid sequences, allowing for detection and visualization purposes.

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6 protocols using cy3 ctp

RNA Extraction and Expression Profiling

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RNA from each of the 69 cell lines was extracted using TRIZOL Reagent following the manufacturer's recommendations (Invitrogen, Burlington, ON). RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Mississauga, ON). RNA quality was assessed by Agilent 2100 Bioanalyzer (Version B.02.02). RNA with an integrity number of at least 7 was amplified and labeled using the Agilent Low RNA Input Linear Amplification kit (Agilent, Santa Clara, CA). Labeling reactions were performed with 250 ng total RNA, along with the Agilent Spike-in RNA mix, using Cy3-CTP and Cy5-CTP for control (-IR) and experimental (+IR) RNA, respectively (Perkin Elmer, MA, USA). Amplified RNA was quantified using the NanoDrop ND-1000 (NanoDrop Technologies, DE, USA) and the concentration of cRNA and the specific dye activity were calculated. Samples with a specific dye activity greater than 8 pmol/µl were selected for hybridization to arrays. Pairs of cRNA (unirradiated versus irradiated) were hybridized to Agilent Whole Human Genome Oligo 4x44K GE arrays as per the product protocol. Image acquisition and analysis were done using an Agilent Microarray Scanner, Model G2565BA and Agilent Feature Extraction software v9.1 set to default settings. Raw data has been submitted to the NCBI GEO database (Accession Number GSE19541.)

Feilotter H.E., Michel C., Uy P., Bathurst L, & Davey S. (2014). BRCA1 Haploinsufficiency Leads to Altered Expression of Genes Involved in Cellular Proliferation and Development. PLoS ONE, 9(6), e100068.

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Agilent Microarray-Based Transcriptomic Analysis

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RNA samples were analyzed for expression profiling using Agilent G3 Human Gene Expression 8 × 60 k Arrays (Agilent; Santa Clara, CA, USA). Total RNA quality for both microarray analyses was determined on an Agilent 2100 Bioanalyzer, with all samples having RNA integrity numbers greater than 8. Total RNA (250 ng) was used for labeling using a QuickAmp labeling kit (Agilent). Extracted RNA was labeled with cyanine (Cy)-3-CTP (PerkinElmer; Waltham, MA, USA) and reference RNA with (Cy)-5-CTP. Following purification of labeled cRNAs, 825 ng of Cy3- and Cy5-labeled cRNAs were combined and hybridized for 17 h at 65 °C in an Agilent hybridization oven. Microarrays were then washed and scanned using an Agilent DNA Microarray Scanner. The mRNA expression profiles in SAEC and HMVEC couture and monoculture are available in NCBI GEO with the accession number GSE129640.

Snyder-Talkington B.N., Dong C., Singh S., Raese R., Qian Y., Porter D.W., Wolfarth M.G, & Guo N.L. (2019). Multi-Walled Carbon Nanotube-Induced Gene Expression Biomarkers for Medical and Occupational Surveillance. International Journal of Molecular Sciences, 20(11), 2635.

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Mouse Genome Microarray Analysis

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Samples were amplified and labeled with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) using Cy3-CTP or Cy5-CTP (Perkin Elmer). Samples were hybridized to Agilent 44 k Mouse Whole Genome Arrays (part no. G4122F) and scanned using an Agilent DNA Microarray Scanner. Amplification, labeling and scanning were carried out as described (58 (link)). Expression profiles were analyzed using single channel intensity analysis, using the LIMMA package in R (59 (link),60 ). Array channels were normalized first between and then within arrays using LIMMA with the ‘quantile’ method. Pairwise comparisons between groups were made using LIMMA. At each time point, WT animals were compared to KO animals, and expression profiles of WT animals at day 1, day 4 and day 14 were compared to those of the unoperated WT animals to determine the regulation after axotomy in WT animals. For all comparisons, a fold-change cut-off of 1.5 was used. A FDR, calculated using the Benjamini-Hochberg method (61 ), of 0.01 was used.

Mason M.R., van Erp S., Wolzak K., Behrens A., Raivich G, & Verhaagen J. (2021). The Jun-dependent axon regeneration gene program: Jun promotes regeneration over plasticity. Human Molecular Genetics, 31(8), 1242-1262.

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Mouse and Human Microarray Expression Analysis

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In vivoexpression profiling of the 160 mouse lung samples was analyzed using Agilent Mouse Whole Genome Arrays as previously reported (Guo et al. 2012 ).In vitro extracted RNA from the 72 cell samples was analyzed for expression profiling using Agilent G3 Human Gene Expression 8 × 60k Arrays (Agilent, Santa Clara, CA). Total RNA quality for both microarray analyses was determined on an Agilent 2100 Bioanalyzer, with all samples having RNA integrity numbers greater than 8. Total RNA (250ng) was used for labeling using a QuickAmp labeling kit (Agilent). Extracted RNA was labeled with cyanine (Cy)-3-CTP (PerkinElmer, Waltham, MA) and reference RNA with (Cy)-5-CTP. Following purification of labeled cRNAs, 825 ng of Cy3-and Cy5-labeled cRNAs were combined and hybridized for 17 h at 65°C in an Agilent hybridization oven. Microarrays were then washed and scanned using an Agilent DNA Microarray Scanner.

Snyder-Talkington B.N., Dong C., Zhao X., Dymacek J., Porter D.W., Wolfarth M.G., Castranova V., Qian Y, & Guo N.L. (2014). Multi-walled carbon nanotube-induced gene expression in vitro: concordance with in vivo studies. Toxicology, 328, 66-74.

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Bovine Gene Expression Microarray Analysis

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Bovine V2 Oligo 4×44 K microarrays (Agilent Technologies, Inc.) were used to determine the differential gene expression between infected and control cells. For reverse transcription, second-strand cDNA was synthesized from 0.5 µg total RNA using the Fluorescent Linear Amplification kit containing T7 RNA polymerase (Agilent Technologies, Inc.). The cDNA served as template for in vitro transcription to produce target cRNA labeled with Cy3-CTP (to label infected cells) and Cy5-CTP (to label control cells) (PerkinElmer, Inc., Waltham, MA, USA). Labeled cRNA (0.825 µg) was fragmented (mean size, ~50–100 nucleotides) in fragmentation buffer (Agilent Technologies, Inc.) at 60°C for 30 min. The prepared cRNA was subsequently hybridized to the microarray at 60°C for 17 h. Two replicates of the microarray assays (M1 and M2) were performed. Hybridized microarray chips were scanned using the Agilent Microarray Scanner with Feature Extraction software 9.5.3 (Agilent Technologies, Inc.). The locally weighted linear regression method was applied to normalize the results by rank consistency filtering.

CHENG Y., CHOU C.H, & TSAI H.J. (2015). In vitro gene expression profile of bovine peripheral blood mononuclear cells in early Mycobacterium bovis infection. Experimental and Therapeutic Medicine, 10(6), 2102-2118.

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Transcriptomic Analysis of ATRA Treatment

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Total RNA was isolated from the cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) at 0, 0.5, and 1 h after the ATRA treatment. The quality of the extracted RNA was checked using a Bioanalyzer 2100, RNA 6000 Nano LabChips, and the 2100 Expert standard software (all Agilent Technologies, Santa Clara, CA, USA). To prepare cDNA, approximately 0.5 g of each RNA sample was used in the reaction of the reverse transcription, performed using the Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA) according to standard protocol. The cRNA samples for time points 0, 0.5, and 1 h after the ATRA treatment were labeled with Cy5-CTP (Perkin Elmer, Waltham, MA, USA) and the control samples (the time point 0 h) were labeled with Cy3-CTP (Perkin Elmer, Waltham, MA, USA). The cRNA fragmentations and hybridizations were performed using in situ the Hybridization Kit Plus (Agilent Technologies, Santa Clara, CA, USA) according to standard protocol. Data acquisition was performed using the DNA Microarray Scanner G2505C (Agilent Technologies, Santa Clara, CA, USA). The raw transcriptome data were processed using the Feature Extraction software (version 10.1.3.1; Agilent Technologies, Santa Clara, CA, USA).
Statistical data analysis by ANOVA with the p-value cut-off set at 0.05 was carried out using the GeneSpring GX12.5 software (Agilent Technologies, Santa Clara, CA, USA).

Novikova S., Tolstova T., Kurbatov L., Farafonova T., Tikhonova O., Soloveva N., Rusanov A., Archakov A, & Zgoda V. (2022). Nuclear Proteomics of Induced Leukemia Cell Differentiation. Cells, 11(20), 3221.

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