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| Status |
Public on Apr 10, 2024 |
| Title |
Head and Neck Squamous cell tumor, Drug+XRT_Wk1_miRNA58 |
| Sample type |
RNA |
| |
|
| Source name |
FaDu human squamous cell carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
tumor type: Head and Neck Squamous cell tumor
tumor origin: hypopharyngeal
mouse strain: Athymic nude mice (nu/nu)
biological sex: Female
age of mice: 6 weeks old
treatment: Drug+XRT
time: wk1
|
| Treatment protocol |
Six-week old Athymic nude nu/nu female mice were injected with FaDu cells in right flank. FaDu cells were allowed to grow to tumor size 200mm3 before treatment started. Mice were divided into 4 groups beyond the initial Control on D1: Untreated (UT), x-ray irradiation only (Rad), Onalespib drug treated only (Drug), and both (Rad + Drug). Mice received i.p injections 5 days per week of control vehicle or Onalespib. Mice received Drug through week 1 and week 2. Mice in the Drug+Rad group had a second week of Drug. In animals receiving Onalespib and radiation, animals were injected with Onalespib 2-3 hours prior to radiation. Mice in Rad groups received 6Gy total radiation 2Gy every other day (2Gy x 3) for the first week. Mice receiving treatments were euthanized at the end of week 1 and at the end of week 2. Animals receiving radiation were euthanized approximately 4 hours post radiation. Control mice were euthanized at the beginning of treatment (D1), and Untreated (vehicle only) at the end of week 1 and Day 8 of treatment schedule due to excessive tumor size. Tumor samples were analyzed at Day 1 at end of Week 1 and Week 2 (indicated in figure 1A, below). Additional mice were followed for tumor growth delay. Longer term tumor samples were not available as this was not the primary purpose of this study.
|
| Growth protocol |
The human squamous cell carcinoma from the pharynx (FaDu) was used for this study. Prior to injection FaDu cells were cultured in DMEM, 10% FBS. These cells were injected into right flank of athymic mice at a concentration of 1 x 10^6 cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor samples were bathed in liquid nitrogen and pulverized into a fine powder using a mortar and pestle. Approximately 100 µg of powdered sample was lysed with 700 µl of QIAzol lysis buffer (Cat # 79306, QIAGEN) and homogenized by passing the solution through QIAshredder spin columns (Cat # 79654, QIAGEN). RNA isolation was performed using standard miRNeasy mini kits (Cat # 217004, QIAGEN) according to the manufacturer’s protocol. Quality and quantity of the RNA samples were assessed using a DeNovix DS-11 nanodrop spectrophotometer (DeNovix, DE, US) and Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA). by spectrophotometry and on the Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
mRNA: Labeled cRNA was prepared from total RNA samples. Briefly, the Poly(A)+ RNA population within total RNA was amplified using MessageAMP II (Applied Biosystems, Foster City, CA). After a second round of reverse transcription, second-strand cDNA synthesis, and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase in the presence of Biotin-11-UTP. The quantity and quality of the cRNA was assayed by spectrophotometry and on the Agilent Bioanalyzer. miRNA: Total RNA samples were dephosphorylated, denatured, and end-labeled with Cy-3.
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| |
|
| Hybridization protocol |
mRNA: 1 µg of purified cRNA was fragmented to uniform size and applied to Agilent Human 8x60 (v3) Gene Expression Microarrays Design ID 072363 (Agilent Technologies, Santa Clara, CA) in hybridization buffer. Arrays were hybridized at 65° C for 17 hrs. in a rotating incubator, washed at 42° C for 30 min, and stained with Streptavidin-Alexa555. miRNA: labeled target was applied to Agilent Human miRNA 8x60 v21.0 arrays (design ID 070156) in hybridization buffer. Arrays were hybridized at 55° C for 20 hrs in a rotating incubator, washed at 37° C for 10 min.
|
| Scan protocol |
Rinsed and dried arrays were scanned with an Agilent G2505C Microarray Scanner (Agilent Technologies, Santa Clara, CA) at 5 µm resolution
|
| Description |
miRNA 1 week post Onalespib (5 days, 55mg/kg) and radiation treatment (3 x 2 Gy)
|
| Data processing |
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
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| |
|
| Submission date |
Apr 20, 2023 |
| Last update date |
Apr 10, 2024 |
| Contact name |
Michelle Bylicky |
| E-mail(s) |
[email protected]
|
| Organization name |
National Cancer Institute
|
| Department |
Radiation Oncology Branch
|
| Street address |
10 Center Drive
|
| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20879 |
| Country |
USA |
| |
|
| Platform ID |
GPL21576 |
| Series (1) |
| GSE230151 |
Cancer treatment with radiation-chemotherapy rapidly induces molecular pathways for both resistance and new therapeutic targets |
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