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Antiangiogenic Antithrombin

ABSTRACT: Confluent passage-2 HUVECs in Falcon T12.5 flasks grown in the presence or absence of VEGF-165 (10ng/mL) (R&D systems) were treated with human native, cleaved or latent antithrombins (20 5g/mL) for 24 hours in 3 mL M-199 with 2% heat-inactivated FBS and antibiotics. Following this treatment, total RNA was extracted from the cells by the Trizol method (Invitrogen). The total RNA was subjected to one cycle of linear RNA amplification using the MessageAmp aRNA kit according to the manufacturers instructions (Ambion, Austin, TX). The quality of the antisense RNA was verified on a denaturing agarose gel. Only samples showing a distribution of sizes from 250-5500 nt with a peak centered at 1000-1500 nt were used to generate the test cDNA samples for the subsequent array experiments. DNA labeling and hybridizations were performed essentially as described (Pollack et al., 1999, Nat. Genet.), with slight modifications. Briefly, the test endothelial cell antisense RNA samples together with Universal Human Reference RNA (Stratagene, Cedar Creek, TX) were used to generate Cy3/Cy5 (Amersham Biosciences) labeled cDNA for array hybridization on the Stanford 43K human cDNA microarray (www.microarray.org/sfgf). The Stanford microarray used in this study consists of 18,416 named genes with UniGene symbol, 4,145 ESTs with known function, 19,365 ESTs with unknown function and ~1,000 repeated spots as internal controls. Hybridized arrays were scanned on a GenePix 4000B scanner (Axon Instruments), and fluorescence ratios (test/reference) calculated using the Stanford Microarray Database software (available at http://genomewww5. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment Keywords: Computed

ORGANISM(S): Homo sapiens

PROVIDER: GSE3956 | GEO | 2005/12/31

SECONDARY ACCESSION(S): PRJNA94209

REPOSITORIES: GEO

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Project description:Background: With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes the development of this method and its application to mapping transcriptome profiles. Results: RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. In addition, five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were artifacts. Conclusions: An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is more sensitive in detecting small amount of RNA compared to end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.

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Antiangiogenic Antithrombin

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