|
| Status |
Public on Sep 11, 2018 |
| Title |
Temsirolimus (iv) followed by Sorafenib (po) in Bone Marrow for 24 hr(s) (6M71) |
| Sample type |
RNA |
| |
|
| Source name |
Temsirolimus (iv) followed by Sorafenib (po)
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
Sex: Male
age: 7-8 weeks
tissue: Bone Marrow
compound given 1st: Temsirolimus
dose (mg/kg) compound given 1st: 0.3
compound given 2nd: Sorafenib
dose (mg/kg) compound 2nd: 25
time: 24 hr
|
| Treatment protocol |
At 1, 6, or 24 hours after the completion of dosing, rats were euthanized by CO2 asphyxiation. Immediately after euthanasia, tissues of interest were collected for RNA isolation. Bone marrow (left femur), heart (apex, with left and right ventricles), liver (left lateral and median lobes). Samples of the heart and liver collected for RNA isolation were cut into sections ≤ 2 mm in thickness and immediately placed into pre-filled tubes containing RNAlater® (Applied Biosystems; Foster City, CA). Bone marrow was flushed from bone using RNAlater®. After collection into RNAlater®, tissues were maintained refrigerated (approximately 4-5 °C) for at least 24 hours and then stored at or below -20 °C.
|
| Growth protocol |
Rats used in this study were procured with an indwelling femoral vein cannula. Prior to dosing on day 1, the catheter of each rat was checked for patency. Rats with patent catheters were randomly assigned to one of 8 treatment groups. On day 1, the rats weighed between 197.6–274.4 grams. Teklad Certified Rodent Diet 2016C (Harlan; Madison, WI) and tap water (Birmingham public water supply) were provided ad libitum to the rats prior to and throughout the study. The animals were individually housed in solid-bottom polycarbonate cages on stainless steel racks in a room maintained at a temperature of 70-79°F and a relative humidity of 44-66%. Room lights were controlled by an automatic timer set to provide a 12/12 light:dark cycle
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation from individual tissues was accomplished using an RNeasy microarray kit (QIAGEN Inc.). After extraction, the RNA concentration of each sample was determined using a RiboGreen assay. The RNA Integrity Number (RIN) of each sample was determined using an Agilent 2100 Bioanalyzer with 2100 Expert Software. Only samples with a RIN of 7.5 or higher were deemed acceptable for gene expression analysis.
|
| Label |
Biotin
|
| Label protocol |
Total RNA samples were converted into labeled target antisense RNA (cRNA) using the Single-Round RNA Amplification and Biotin Labeling System (Enzo Life Sciences, Farmingdale, NY).
|
| |
|
| Hybridization protocol |
11 μg of purified cRNA was applied to Affymetrix GeneChip Rat Genome 230 2.0 Arrays, and incubated at 45°C for 16 hours. Following hybridization, arrays were washed and stained using standard Affymetrix procedures.
|
| Scan protocol |
Arrays were scanned on the Affymetrix GeneChip Scanner 3000 using factory PMT settings. Data extraction was completed with Expression Console software using a target scaling of 500.
|
| Description |
The rat was administered a single bolus intravenous (iv) dose of temsirolimus or appropriate vehicle formulation. Thirty (30) minutes after iv administration of temsirolimus or vehicle formulation, sorafenib, sunitinib, or respective vehicle formulation was administered po. IV doses were administered in a dose volume of 5 mL/kg. PO doses were administered in a dose volume of 10 mL/kg. Dose volumes were based on the most recent body weight taken.
|
| Data processing |
Data analysis was performed using the Bioconductor R package using the affyQCReport, simpleaffy and arrayQualityMetrics packages. The raw data was normalized using the robust multichip average (RMA) algorithm (Irizarry et. al. Biostatistics, 2003) with log2 transformation.
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| |
|
| Submission date |
Aug 28, 2018 |
| Last update date |
Sep 11, 2018 |
| Contact name |
Myrtle Davis Millin |
| Organization name |
The National Cancer Institute
|
| Street address |
31 Center Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1355 |
| Series (2) |
| GSE119122 |
Transcriptomic profiles of tissues from rats treated with drug combinations [Study 1] |
| GSE119135 |
Transcriptomic profiles of tissues from rats treated with drug combinations |
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