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.
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.