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Sample GSM1371287 Query DataSets for GSM1371287
Status Public on Jan 07, 2015
Title HOC182_pt
Sample type RNA
 
Source name Ovarian cancer from patient
Organism Homo sapiens
Characteristics cell type: Epithelial Ovarian Cancer (EOC)
tissue: patient-derived tumor
Treatment protocol EOC-xenografts collected from subcutis (sc), abdominal masses (am) and ascitic fluid (A) of mice engrafted with tumors at different passages (62 xenograft samples representing 29 EOC-xenograft models) and from 9 patient specimens, underwent one-color microarray-based gene expression profiling by way of SurePrint G3 Human GE V2 8x60K microarrays (50,599 Biological Features/array; Agilent Technologies) .
Growth protocol EOC-xenografts Subcutis models (s.c.). Tumors were dissected free of necrotic tissue repeatedly rinsed in HBSS and 2-4 mm of tissue was implanted subcutaneously in the flank of nude mice. Tumor growth was measured with a Vernier caliper, and tumor weights (mg = mm3) were calculated as follows: (length [mm] × width2 [mm2]) / 2. Intraperitoneal models (i.p.). Ascites was centrifuged, washed repeatedly, resuspended in HBSS and implanted intraperitoneally in nude mice at a concentration of 10-20 ×10e6. Criteria for growing tumors were abdominal distension and palpable tumor masses in the peritoneal cavity. Mice were killed when they presented signs of discomfort (survival), ascites was harvested and the volume recorded. Intrabursal transplantation (i.b.). Ovarian cancer cells from enzymatic digestion or ascites (1×10e6 cell suspension) were injected orthotopically under the bursa of the mouse ovary, as detailed in Supplementary data. At necroscopy, the ovary image was acquired with a macrodigital imaging system (MacroPATH; Milestone S.r.l, Sorisole, Italy); the two diameters were determined, the mean calculated and taken as measure of tumor mass. Ascites was harvested and the volume recorded. Clinical specimens (primary ovarian tumors, metastasis, ascitic fluid) were obtained from patients undergoing surgery for ovarian tumor by laparotomy or paracentesis and in nude mice within 24hr. Established EOC-xenografts were cryopreserved frozen in DMSO and passed serially in nude mice for further studies (i.e. EOC-xenograft sample were immediately snap frozen for gene expression analysis).
Extracted molecule total RNA
Extraction protocol RNA isolation and assessment of human and mouse proportion. Qiazol and the miRNeasy Mini Kit together with a DNase I digestion step (Qiagen) were used according to the manufacturer’s recommendations to isolate total RNA from EOC-xenografts and patient tumor specimens. Total RNA quality was checked for integrity using RNA Nano Chips on Agilent Bioanalyzer 2100 (Agilent Technologies). Xenograft tumors are composed of a mixture of human- and mouse-derived cells; to assess their respective amount, total RNA was evaluated by way of species-specific TaqMan Prime Time qPCR assays for beta actin (ACTB). Briefly, 200ng tot RNA were reverse transcribed with random hexamers in a 20 microliters reaction by the High Capacity cDNA Reverse Transcription kit (Applied Biosystems); cDNA (2 microliters of the 1:20 dilution) was PCR amplified in 10 μl reaction, in presence of 500nM primers, 250nM probe (Hs PT39a22214847, MmPT53a.31778008, IDT) and the TaqMan gene expression master mix 1x (Applied Biosystem) on the Applied Biosystems 7900HT Fast Real Time PCR system (40 cycles at 94°C for 15 s, 60°C for 60s). Standard curves, generated with the following formula: [CtmouseACTB – CthumanACTB = (m-h Ct)] using known using known human and mouse tot RNA mixtures (100%, 95, 90 ,80, 75, 70, 60, 50%), were used to determine the proportions of human and mouse ACTB RNA in EOC-xenograft. The value (m-h Ct) obtained for each sample (analyzed in triplicate) was plotted against the standard curve to extrapolate the percentage of human RNA. Only samples with a human RNA content > 75% were considered further.
Label Cy3
Label protocol Labeled cRNA target was prepared starting from100 ng of input RNA [RNA integrity number (RIN) > 7.0] by Low Input Quick Amp Labelling Kit and One-Color Spike-In Kit (Agilent Technologies) and purified with RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendation and protocols. Cyanine3CTP-labelled cRNA was quantified on a NanoDrop ND1000 Spectrophotometer using Microarray/RNA-40 measurement and the specific activity of cyanine 3 was calculated for each reaction. We set the minimum threshold of 2 μg for yield and 7 pmol/μg for the specific activity. Labeled cRNA (600ng) was fragmented by incubation with 5μl of 10× blocking reagent and 1μl of 25× fragmentation buffer in a 25μl reaction volume for 30 minutes at 60°C. Twentyfive microliters of 2× GE Hybridization Buffer Hi-RPM was added to fragmented cRNA; 40 μl of this “hybridization mix” were placed onto the SurePrint G3 Human GE V2 8x60K microarrays (50,599 Biological Features/array; Agilent Technologies).
 
Hybridization protocol Hybridization was carried out for 17 hours at 65°C by rotating at 10 rpm. Microarray slides were washed in GE Wash Buffer-1 for 2 minutes and pre-warmed GE Wash Buffer-2 for 2 minutes at room temperature.
Scan protocol Microarray slides were scanned in an Agilent Scanner version C (G2505C, Agilent Technologies).
Data processing Images were analyzed using the Feature Extraction software v10.7. Raw data elaboration was carried out with Bioconductor (www.bioconductor.org), using R statistical language. Background correction was performed with the normexp method with an offset of 50 and quantile was used for between-array normalization. The LIMMA (LInear Models for Microarray Analysis) package was then used to identify differentially expressed genes in EOC-xenografts versus their corresponding patient tumors. The empirical Bayes method was used to compute a moderated t-statistics. Transcripts with a log base two fold change (logFC) greater than 1 or lower than -1 and p-value less than 0.01 were considered as differentially expressed.
 
Submission date Apr 18, 2014
Last update date Apr 23, 2018
Contact name giovanna chiorino
E-mail(s) giovanna.chiorino@gmail.com
Organization name Fondo Edo Tempia
Department Cancer Genomics
Street address via malta 3
City Biella
ZIP/Postal code 13900
Country Italy
 
Platform ID GPL17077
Series (1)
GSE56920 A platform of patient-derived tumor xenografts (EOC-xenografts) to recapitulate the clinicopathology and genetic alterations of ovarian cancer
Relations
Reanalyzed by GSE113533

Data table header descriptions
ID_REF
VALUE Log2 Intensity values after background correction and quantile normalization

Data table
ID_REF VALUE
A_23_P117082 11.69808871
A_33_P3246448 7.660459376
A_33_P3318220 6.395127378
A_33_P3236322 6.264010255
A_33_P3319925 7.178561449
A_21_P0000509 17.25873848
A_21_P0000744 9.745408724
A_24_P215804 5.888733172
A_23_P110167 12.95107664
A_33_P3211513 9.384616505
A_23_P103349 6.445986126
A_32_P61480 6.561596586
A_33_P3788124 6.343790356
A_33_P3414202 9.8766404
A_33_P3316686 8.102821022
A_33_P3300975 10.77498155
A_33_P3263061 11.93744946
A_33_P3261373 6.467854809
A_24_P278460 8.115346025
A_21_P0013109 6.411935571

Total number of rows: 50599

Table truncated, full table size 1254 Kbytes.




Supplementary file Size Download File type/resource
GSM1371287_US22502614_253949410390_S02_GE1_107_Sep09_1_2.txt.gz 12.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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