|
| Status |
Public on Oct 05, 2022 |
| Title |
PDX_HCI_005_ESR1_L536P_miRNA_expression |
| Sample type |
RNA |
| |
|
| Source name |
mammary gland ductal carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
model: HCI-005 PDX
esr1 genotype: L536P
|
| Treatment protocol |
ER mutant or WT T-47D cells were plated in 100mm dishes and grown in estrogen-deprived media for four days prior to treatment with DMSO (0.1%) for 8 hours. For ESR1 knock-down experiments, cells were treated with DMSO (0.1%) and transfected with siRNAs targeting ESR1 or a non-targeting siRNA control for 48 hours prior to RNA harvest. PDX samples were surgically removed from mice and flash frozen. PDX samples were not treated.
|
| Growth protocol |
Cells were cultured in RPMI1640 Media (ThermoFisher Scientific) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (ThermoFisher Scientific).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed with phosphate-buffered saline (PBS; ThermoFisher Scientific) and treated with RLT plus (Qiagen) lysis buffer with 1% beta-mercaptoethanol (BME; Sigma-Aldrich) for cell lysis. Cells were passed through a 21-gauge needle (Sigma-Aldrich) to facilitate genomic DNA lysis. For PDX lines, tissue samples were used from seven PDX lines (WT: HCI-003 and HCI-011; L536P: HCI-005, HCI-006, HCI-007; Y537S: HCI-013, HCI-013EI). Approximately 20mg of tissue per sample was homogenized in RLT plus lysis buffer with 1% beta-mercaptoethanol using gentleMACS M-tubes and a gentleMACS Octo Dissociator (Miltenyi Biotec). RNA extraction for both T-47D and PDX samples was performed using a Quick-RNA RNA Miniprep kit (Zymo Research), and RNA was measured using a Qubit fluorometer.
|
| Label |
NanoString
|
| Label protocol |
Samples were prepared per manufacturer's instructions using the NanoString human v3 miRNA panel (Catalog # CSO-MIR3-12).
|
| |
|
| Hybridization protocol |
Hybridization of nCounter Reporter Probes was performed per manufacturer's instructions using the NanoString nCounter platform with the human v3 miRNA panel.
|
| Scan protocol |
Sample scans were performed on the nCounterTM Digital Analyzer (NanoString Technologies).
|
| Data processing |
NanoString miRNA expression assay results in the form of raw counts were analyzed using NanoString’s nSolver software. Raw counts were normalized using two approaches. The first approach used the geometric mean for each sample of the top 100 highest expressed miRNAs across all samples to determine a normalization factor for each sample. The second approach used the geometric mean for each sample of the five spike-in miRNAs profiled for the assay to generate the normalization factors. Normalized counts were then log-scaled and used to perform t test to identify significantly differentially expressed miRNAs.
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| |
|
| Submission date |
Sep 30, 2022 |
| Last update date |
Apr 19, 2023 |
| Contact name |
Jason Gertz |
| E-mail(s) |
jay.gertz@hci.utah.edu
|
| Organization name |
University of Utah
|
| Lab |
Gertz
|
| Street address |
1950 Cir of Hope Dr
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL32712 |
| Series (2) |
| GSE214578 |
Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs (NanoString) |
| GSE214580 |
Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs |
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