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Sample GSM818971 Query DataSets for GSM818971
Status Public on Oct 01, 2012
Title Day 3 - AM - Bioreactor Run 3 - 5L - mAdbID:106656
Sample type RNA
 
Channel 1
Source name Universal Mouse Reference
Organism Mus musculus
Characteristics reference: All-inclusive universal mouse reference RNA pool (Becton, Dickinson, and Co., Franklin Lakes, NJ).
Extracted molecule total RNA
Extraction protocol Becton-Dickinson Extraction Protocol
Other: not available
Label cy3
Label protocol Cy3 Labeling Protocol
Other: After reverse transcription of mRNA to cDNA, the cDNA was purified by removing the remaining t-RNA and r-RNA using the Minelute (Qiagen) kit. The probe was eluted using 12 µL of elution buffer. 5µL of coupling buffer(0.2M NaHCO3, pH 9) was added to each sample, and fluorescently labeled with 15 µL of Cy3 dye for the reference sample, and incubated in the dark for 90 minutes. Residual dye was removed afterwards using the same MinElute PCR Purification Kit. Following incubation, the probe was cleaned up to remove residual dye using the Qiagen MinElute kit.
 
Channel 2
Source name Day3-AM -5L
Organism Mus musculus
Characteristics cell line: A Sp2/0 myeloma based mouse hybridoma cell line (2055.5) secreting an anti-meningitidis-capsular-polysaccharide (anti-MCPS) IgG3 (Rubinstein LJ 1988) was used as the experimental cell line.
Treatment protocol Treatment time: 67-69 hours
In-vitro treatment: For the experiments reported here, each of the biological replicates had cells thawed and passaged for 6-8 passages prior to 5L experimentation. The 5L bioreactor with 4.5L working volume was inoculated at 2.0±0.5 x 105 cells/mL. Samples were taken for microarray analysis twice daily, approximately 12 hours apart, at the following time-points for all biological replicates - day 1am (18-20 hours), day 2am (42-44 hours), day 3am (67-69 hours) and day 3pm (78-80 hours). It is to be noted that the last sample used for microarrays was obtained when the culture viability was >80% viability. The samples were used for measuring (1) cells count and viability using a hemocytometer with trypan blue dye (2) ELISA (3) glucose and lactate concentration and (4) Cell pellet for microarrays
Extracted molecule total RNA
Extraction protocol Trizol Extraction Protocol
Other: Total RNA from the cells was extracted with Trizol (Gibco Brand, Carlsbad, CA) and chloroform (Mallinckrodt, Phillipsburg, NJ), and purified using the RNeasy Mini-Prep Kit (Qiagen Inc., Valencia, CA).
Label cy5
Label protocol HyPer5 Labeling Protocol
Other: After reverse transcription of mRNA to cDNA, the cDNA was purified by removing the remaining t-RNA and r-RNA using the Minelute (Qiagen) kit. The probe was eluted using 12 µL of elution buffer. 5µL of coupling buffer(0.2M NaHCO3, pH 9) was added to each sample, and fluorescently labeled with 15 µL of HyPer5 dye(Amersham, Piscataway, NJ) for the test samples, and incubated in the dark for 90 minutes. Residual dye was removed afterwards using the same MinElute PCR Purification Kit. Following incubation, the probe was cleaned up to remove residual dye using the Qiagen MinElute kit.
 
 
Hybridization protocol Hybridization Protocol
Other: Polylysine coated glass slides with 36,055 mouse genome chip from Operon were used. These slides included some repeat spots for internal control and were obtained from NCI/CBER microarray facility (Bethesda, MD). The Maui mixing chamber (Biomicro, Salt Lake City, UT) was used for hybridization, with flow-volume of 42 µL. Ambion hybridization buffer was pre-heated to 42oC and 24µL of the buffer was added to 24µL of the clean coupled probe, and injected into the Maui hybridization chamber (Biomicro). The slide was incubated overnight at 42oC, washed with 1X SSC buffer. After the overnight hybridization period, the MAUI mixers were disassembled and the arrays were washed in series with 1X SSC (0.05% SDS) and 0.1X SSC, each for 4 minutes.
Scan protocol Creator: GenePix Pro 5.1.0.16
Scanner: GenePix 4000B [82720]
ScanPower: 100;; 100
LaserPower: 3.07;; 1.86
Temperature: 28.74
Scanning Protocol
Other: The arrays were then scanned for Cy3 and Hyper5 fluorescent emission intensities using a GenePix 4000B microarray scanner (Axon, Union City, CA) with 5um resolution. Image processing, consisting of automatic and manual spot selection/rejection, and spot identification (gridding) was carried out using GenePix Pro 6.0 software (Axon). Data files were generated containing the gene represented by each spot on the array and the spot's corresponding Cy3 and Hyper5 intensities.
Description mAdb experiment ID: 106656
Data processing Data Processing Protocol
Calculation Method: A total of 14589 genes were found to be expressed in the samples tested. This was based on signal filter of >100, and signal to background above 1 standard deviation, as well as size filter for the spots, along with the spot mapping to GAL (NCI Production Gene Array List Files, NIH) files. Lowess normalization (Yang, 2002) was used on the raw data to correct for intensity based bias in 2-color data. In addition 25 bad or not-found genes were filtered out and omitted from gene analysis to avoid false positives.
 
Submission date Oct 18, 2011
Last update date Oct 01, 2012
Contact name Bhargavi Kondragunta
E-mail(s) bhargavi_sk@yahoo.com
Organization name University of Maryland Baltimore County
Department Chemical and Biochemical Engineering
Street address 1000 Hilltop circle
City Baltimore
State/province MD
ZIP/Postal code 21250
Country USA
 
Platform ID GPL14743
Series (2)
GSE33062 Genomic analysis for predicting cell culture status in a sparged bench-scale 5L bioreactor
GSE33063 Process-sensitive sentinel genes as novel cell culture comparability metrics

Data table header descriptions
ID_REF mAdb well id plus replicate number
VALUE log ratio (log2 of CY5 channel/CY3 channel)

Data table
ID_REF VALUE
6322047_1
6324361_1
6341255_1
6341556_1
6331875_1
6340683_1
6340801_1 4.771
6326683_1
6324380_1
6329333_1 4.627
6328399_1 4.098
6335482_1 3.855
6319563_1 4.253
6325333_1
6340588_1 3.989
6322173_1 3.956
6336407_1 4.379
6338384_1
6308930_1 4.191
6332090_1 3.645

Total number of rows: 15847

Table truncated, full table size 203 Kbytes.




Supplementary file Size Download File type/resource
GSM818971_106656.gpr.gz 3.6 Mb (ftp)(http) GPR
Processed data included within Sample table

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