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| Status |
Public on Aug 06, 2015 |
| Title |
C6 glioma xenografts, (R,R’)-4-methoxy-1-naphthylfenoterol (MNF) treated, replicate #4, cohort #2 |
| Sample type |
RNA |
| |
|
| Source name |
The rat-derived C6 glioma cell line was obtained from the American Type Culture Collection (Manassas, VA).
|
| Organism |
Rattus norvegicus |
| Characteristics |
host strain: SWISS nu+/nu+
tumor cell type: The rat-derived C6 glioma cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were routinely maintained in DMEM supplemented with L-glutamine, 1% penicillin/streptomycin solution, and 10% FBS in a humidified CO2 incubator at 37 ºC.
tumor inoculation: Nude mice were inoculated subcutaneously with 100µl of culture medium containing 0.5 x 106 C6 glioma cells in the left flank.
treatment of mice: 3 days after cell inoculation, mice received daily intraperitoneal injection of MNF (2 mg.kg-1) in 100µM ascorbic acid in saline five days a week for 19 days.
isolation of tumor cells: The mice were monitored up to 19 days after MNF injection or euthanized earlier if the tumor size was superior to 2 cubic cm or the mouse was lethargic, sick and unable to feed, which caused the body weight to drop below 20% of initial weight. The mice were euthanized by cervical extension, and tumor masses were removed, weighed and washed with cold PBS before being snap-frozen in liquid nitrogen
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| Treatment protocol |
Starting 3 days after tumor cell inoculation, mice received daily intraperitoneal injection of either (10 µl.g-1 bw) of vehicle or MNF (2 mg.kg-1) in 100µM ascorbic acid in saline (vehicle) five days a week for 19 days.
|
| Growth protocol |
The rat-derived C6 glioma cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were routinely maintained in DMEM supplemented with L-glutamine, 1% penicillin/streptomycin solution, and 10% FBS in a humidified CO2 incubator at 37 ºC. Athymic female nude mice (SWISS nu+/nu+) were obtained from Charles Rivers (L’Arbresle, France) and maintained under pathogen-free conditions with a 12 h light/12 h dark cycle. Animals were fed ad libitum with normal chow. Nude mice were inoculated subcutaneously with 100µl of culture medium containing 0.5 x 106 C6 glioma cells in the left flank. The mice were monitored up to 19 days after MNF injection or euthanized earlier if the tumor size was superior to 2 cubic cm or the mouse was lethargic, sick and unable to feed, which caused the body weight to drop below 20% of initial weight. The mice were euthanized by cervical extension, and tumor masses were removed, weighed and washed with cold PBS before being snap-frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy kit (Qiagen) according to the manufacturer’s instructions. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.
|
| Label |
Streptavidin-Cy3 bound to biotin labeled cRNA.
|
| Label protocol |
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
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| |
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| Hybridization protocol |
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix Rat Ref-12 v1 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has ~22,000 annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
|
| Description |
Athymic female nude mice (SWISS nu+/nu+) were obtained from Charles Rivers (L’Arbresle, France) and maintained under pathogen-free conditions with a 12 h light/12 h dark cycle. Animals were fed ad libitum with normal chow.
C6 glioma xenografts, (R,R’)-4-methoxy-1-naphthylfenoterol (MNF) treated, replicate #4, cohort #2
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std.
Detection_Pval = Detection Pvalue from Illumina GenomeStudio software, ver 1.6.0.
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| |
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| Submission date |
Mar 19, 2013 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL10239 |
| Series (1) |
| GSE45307 |
Antitumor Activity of (R,R’)-4-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse |
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