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Sample GSM339438 Query DataSets for GSM339438
Status Public on Jan 01, 2009
Title Heps_St_APAP5_rat2
Sample type RNA
 
Channel 1
Source name primary hepatocytes, sandwich-cultured, 5 mM APAP, 24h
Organism Rattus norvegicus
Characteristics Male Wistar (Crl: (WI) WU BR), 9-12 weeks of age, 180-250 g
Treatment protocol After 72h of culture, three independent rat hepatocyte cultures (biological replicates) cultured in two conditions (St. and Mod.) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol Male Wistar rats (Crl: (WI) WU BR), 9-12 weeks of age, were obtained from Charles River GmbH, Sulzfeld, Germany. During the acclimatization period and until sacrifice, animals were housed individually in macrolon cages with wire tops and sawdust bedding at 22 degrees Celsius and 50-60% humidity. The light cycle was 12 h light and 12 h dark. Feed and tap water were available ad libitum. Animal protocols for hepatocyte isolation were approved by the institutional Animal Ethics Committee. Hepatocytes were isolated acording to a two-step collagenase perfusion technique as described by Seglen (Seglen, 1976) with minor modifications. Hepatocyte preparations with viability greater than 85% as determined by Trypan Blue exclusion were used and cultured on collagen gel precoated 6-well plates at a density of 1.3 x 10-E6 cells per well. Sandwich-cultures of rat hepatocytes were essentially prepared according to the method of Beken (Beken et al., 2004) with culture conditions as described by Kienhuis (Kienhuis et al., 2006, Kienhuis et al., 2007). After attachment for 4 h in attachment medium, dead cells were removed by washing and the upper collagen layer was applied. Thereafter, cells were kept in DMEM containing either standard culture medium (St.) consisting of DMEM supplemented with 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 µg/ml hydrocortisone hemisuccinate, and 50 µg/ml gentamycin, or modified culture medium (Mod.) consisting of standard culture medium (St.) supplemented with 1 mM phenobarbital (PB), 10 µM dexamethasone (DEX), and 5 µM β-naphthoflavone (β-NF) (Kienhuis et al., 2007). PB was added as a concentrated stock solution in PBS. DEX and β-NF were added as concentrated stock solutions in DMSO. The final DMSO concentration was equalized in all culture media (St. and Mod.) and did not exceed 0.2% (v/v). Cultures were incubated at 37 degrees Celsius in a humidified incubator gassed with 5% CO-2 in air. Medium was changed on a daily basis during a period of 72 h.
Extracted molecule total RNA
Extraction protocol After 24h exposure time, culture medium of sandwich-cultured hepatocytes was removed, Trizol was added onto the upper collagen layer and cells were collected. RNA was purified using the RNeasy MinElute kit including an additional DNA digestion step. RNA quality was determined using the Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples contained intact total RNA with a 28S/18S rRNA ratio > 1.5 and a RIN number > 8. RNA extractions of three rats cultured in two conditions (St. and Mod.) were used for microarray analysis.
Label Cy5
Label protocol RNA samples from control hepatocyte cultures and cultures exposed to APAP were labeled with cyanine 5-CTP (Cy5). Cy5 labeled samples from one rat were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same rat. As a result, control samples labeled with Cy5 hybridized agains Cy3 samples can be considered a self-self hybridization. Labeling was performed using Agilent's low RNA input fluorescent linear amplification kit followin manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using molony murine leukemia virus-reverse transcriptase (MMLV-RT) with T7 promoter primer, starting with 1 µg of total RNA. Cyanine-labeled cRNA targets were transcribed using T7 RNA Polymerase. The amlified cRNA was purified using RNeasy mini spin columns. Synthesized cRNA products were quantified spectrophotometrically.
 
Channel 2
Source name primary hepatocytes, sandwich-cultured, non-exposed
Organism Rattus norvegicus
Characteristics Male Wistar (Crl: (WI) WU BR), 9-12 weeks of age, 180-250 g
Treatment protocol After 72h of culture, three independent rat hepatocyte cultures (biological replicates) cultured in two conditions (St. and Mod.) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol Male Wistar rats (Crl: (WI) WU BR), 9-12 weeks of age, were obtained from Charles River GmbH, Sulzfeld, Germany. During the acclimatization period and until sacrifice, animals were housed individually in macrolon cages with wire tops and sawdust bedding at 22 degrees Celsius and 50-60% humidity. The light cycle was 12 h light and 12 h dark. Feed and tap water were available ad libitum. Animal protocols for hepatocyte isolation were approved by the institutional Animal Ethics Committee. Hepatocytes were isolated acording to a two-step collagenase perfusion technique as described by Seglen (Seglen, 1976) with minor modifications. Hepatocyte preparations with viability greater than 85% as determined by Trypan Blue exclusion were used and cultured on collagen gel precoated 6-well plates at a density of 1.3 x 10-E6 cells per well. Sandwich-cultures of rat hepatocytes were essentially prepared according to the method of Beken (Beken et al., 2004) with culture conditions as described by Kienhuis (Kienhuis et al., 2006, Kienhuis et al., 2007). After attachment for 4 h in attachment medium, dead cells were removed by washing and the upper collagen layer was applied. Thereafter, cells were kept in DMEM containing either standard culture medium (St.) consisting of DMEM supplemented with 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 µg/ml hydrocortisone hemisuccinate, and 50 µg/ml gentamycin, or modified culture medium (Mod.) consisting of standard culture medium (St.) supplemented with 1 mM phenobarbital (PB), 10 µM dexamethasone (DEX), and 5 µM β-naphthoflavone (β-NF) (Kienhuis et al., 2007). PB was added as a concentrated stock solution in PBS. DEX and β-NF were added as concentrated stock solutions in DMSO. The final DMSO concentration was equalized in all culture media (St. and Mod.) and did not exceed 0.2% (v/v). Cultures were incubated at 37 degrees Celsius in a humidified incubator gassed with 5% CO-2 in air. Medium was changed on a daily basis during a period of 72 h.
Extracted molecule total RNA
Extraction protocol After 24h exposure time, culture medium of sandwich-cultured hepatocytes was removed, Trizol was added onto the upper collagen layer and cells were collected. RNA was purified using the RNeasy MinElute kit including an additional DNA digestion step. RNA quality was determined using the Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples contained intact total RNA with a 28S/18S rRNA ratio > 1.5 and a RIN number > 8. RNA extractions of three rats cultured in two conditions (St. and Mod.) were used for microarray analysis.
Label Cy3
Label protocol RNA samples from control hepatocyte cultures and cultures exposed to APAP were labeled with cyanine 5-CTP (Cy5). Cy5 labeled samples from one rat were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same rat. As a result, control samples labeled with Cy5 hybridized agains Cy3 samples can be considered a self-self hybridization. Labeling was performed using Agilent's low RNA input fluorescent linear amplification kit followin manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using molony murine leukemia virus-reverse transcriptase (MMLV-RT) with T7 promoter primer, starting with 1 µg of total RNA. Cyanine-labeled cRNA targets were transcribed using T7 RNA Polymerase. The amlified cRNA was purified using RNeasy mini spin columns. Synthesized cRNA products were quantified spectrophotometrically.
 
 
Hybridization protocol For microarray hybridization, Cy5-labeled samples and Cy3-labeled samples were combined. cRNAs were fragmented at 60 degrees Celsius for 30 min with fragmentation solution followed by hybridization on Agilent 22K format 60-mer oligo microarrays (G4130A from Agilent Technologies, Palo Alto, CA) for 17h at 60 degrees Celsius with Agilent hybridization solution. Arrays were washed according to manufacturer's instruction.
Scan protocol Microarrays were scanned using a Packard Scanarray Express confocal laser scanner (PerkinElmer, Boston, MA).
Description St_Standard culture medium
Data processing Flagged spots, consisting of poor quality spots and negative and positive control spots, were excluded. For each spot, median local background intensity was subtracted from the median spot intensity and spots from low expression genes (with a net intensity of < 40 in both channels), were excluded from further analysis. These background-corrected median intensities were log transformed base 2. Data were normalized using the Lowess algorithm.
 
Submission date Nov 04, 2008
Last update date Nov 05, 2008
Contact name Anne Susan Kienhuis
E-mail(s) [email protected]
Organization name RIVM
Street address A. van Leeuwenhoeklaan 9
City Bilthoven
ZIP/Postal code 3720 BA
Country Netherlands
 
Platform ID GPL890
Series (1)
GSE13465 Acetaminophen-induced gene expression profiles in sandwich-cultured primary rat hepatoctyes

Data table header descriptions
ID_REF
VALUE Lowess normalized log 2 ratio (Cy5/Cy3); signal calculated using Imagene 5.0 and GeneSight 4.1 software (Biodiscovery, Palo Alto, CA).

Data table
ID_REF VALUE
3 -0.463191636
4 0.652909567
5
6
8 0.403184407
9
10 -0.076343268
11 0.33306177
12 0.320768422
13 0.350506513
15 -0.294347178
16 0.289387039
17 0.010220657
18
19 0.152664908
20
22
23
24
25 -0.625408112

Total number of rows: 20501

Table truncated, full table size 306 Kbytes.




Supplementary file Size Download File type/resource
GSM339438_Cy3_36570.txt.gz 2.0 Mb (ftp)(http) TXT
GSM339438_Cy5_36570.txt.gz 1.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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