Unknown,Transcriptomics,Genomics,Proteomics

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The Study of Gene Expression Profiles of KG-1 Cells Treated with Antrodia Camphorata Extracts using cDNA microarray.

ABSTRACT: Antrodia Camphorata is well known in Taiwan as a folk medicinal fungus with anticancer and anti-inflammatory effects. In this study, we used a human acute myelogenous leukemia cell line, KG-1, as the experimental model and constructed a high-throughput gene screen integrated platform by using a combination of herbal identification, UV-VIS spectrophotometry, high performance liquid chromatography (HPLC), inductively coupled plasma mass spectrometry (ICPMS), and DNA microarray technology. Based on this science-based platform, we developed a practical quantitative method and also established an organic/inorganic chemical fingerprint for the herbal materials. In the mean while, the global gene expression patterns of the KG-1 cells treated with various A. camphorata mycelia extracts (water and methanol extracts) were compared. Furthermore, several possible biological responses and relevant marker gene would be identified by our high-throughput integrated platform. Keywords: KG-1 treated with AC extracts for 4 days, cDNA microarray, biomarker selection. We used optimal loop design to calculate the gene expression profile. In this design, we used labeled RNA from each sample to labeled three slides for a total of nine arrays. Therefore, the data computations were consisted of 18 measurements (6 samples with 3 replicates) for each of 7334 genes. Besides, duplicated water-treatment samples were used as internal control

ORGANISM(S): Homo sapiens

SUBMITTER: ChengWei Chang

PROVIDER: E-GEOD-3692 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In order to study the function of the Campylobacter jejuni Cj1501 gene, a series of experiments were carried out. Three strains were constructed: a Cj1501 knockout strain, a strain where the Cj1501 knockout was complemented in trans, and a strain with a second copy over-expressing Cj1501 from an fdxA promoter. The transcriptomes of these were all compared to the wild-type strain. The arrays are all from RNA isolated in mid-exponential growth from independent biological replicates. Batch cultures of Campylobacter jejuni NCTC 11168 were grown in 50 ml volumes of Brucella broth in 70cm tissue culture flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (5% Oxygen, 10% Carbon dioxide, 85% Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 37 M-BM-:C. RNA was extracted from C. jejuni cultures grown to an OD600 of approximately 0.4. Briefly, 0.1 volume of 5% phenol in ethanol was mixed with the broth culture, and after centrifugation RNA was isolated with Tri Reagent (Sigma) and chloroform. RNA was further purified using the RNeasy kit (Qiagen) according to the manufacturerM-bM-^@M-^Ys instructions. The RNA was treated with Turbo DNA-free (Ambion) to remove any residual DNA according to the manufacturerM-bM-^@M-^Ys instructions. RNA concentration was determined using the Nanodrop Spectrophotometer NS-1000 (Thermo Scientific). Two or three independent RNA preparations (biological replicates) of each sample type were labelled and hybridized to custom-designed Agilent microarrays. Equal quantities of RNA from test and control cultures were labelled by using nucleotide homologues of dUTP containing either Cy3 or Cy5 fluorescent dye (Perkin Elmer). For each microarray slide, the test strain was labelled with Cy3-dUTP, while the wild-type sample was labelled with Cy5-dUTP. RNA (15 M-BM-5g) was primed with 5 M-BM-5g pd(N)6 random hexamers (Amersham Biosciences). The Affinity Script kit (GE Healthcare) was used to produce cDNA, and hybridized (Agilent Hi-RPM Gene Expression Hybridization Kit) to the microarray slide according to the manufacturerM-bM-^@M-^Ys instructions. Microarrays were scanned at 5 M-BM-5m using an Axon 4000A scanner, and images were acquired using GenePix Pro 3.0 software (Axon). Related analysis reference: Holmes K., Mulholland F., Pearson B. M., Pin C., McNicholl-Kennedy J., Ketley J. M., Wells J. M. (2005) Campylobacter jejuni gene expression in response to iron limitation and the role of Fur Microbiology-SGM 151 243-257 (PMID 15632442).

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The Study of Gene Expression Profiles of KG-1 Cells Treated with Antrodia Camphorata Extracts using cDNA microarray.

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