|
| Status |
Public on Apr 04, 2019 |
| Title |
DTC.T1830.50CD45.1 |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow Leukocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cancer type: Breast cancer
tumor stage: Early
gender: Female
source tissue: Bone Marrow
matched leukocyte sample: n.a.
|
| Treatment protocol |
Cell isolation via IE/FACS: If a patient had ≥1 DTC/mL via Immunomagnetic Enrichment/Flow Cytometry (IE/FC) the remaining volume of bone marrow was subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) within 24-32 hr after bone marrow draw. In the initial step, magnetic beads coated with EpCAM mAb are used to enrich for tumor cells. Enriched samples are then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+/CD45-) from leukocytes (CD45+/EpCAM-) during sorting. This procedure allows for the isolation of small pools of cells, which can be subjected to downstream molecular analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were sorted into reaction tubes containing 9.5µL of lysis buffer and 0.5 µL of DNAse. Lysis reaction was carried out for 5 min at room temperature and 1µL of Stop solution was added and incubated for 2 min and immediately placed on ice. Whole cell lysates were then stored in -80°C until further use.
|
| Label |
FAM
|
| Label protocol |
Reverse transcription (RT) and preamplification were performed using Taqman® PreAmp Cells-to-CT™ kit (Ambion, Texas) following manufacturer’s instructions with some modification. These steps were performed in the same tube the cells were sorted into to avoid any loss of nucleic acids. RT was performed on total RNA in the whole cell lysate (11µL) by adding 12.5µL of 2X RT buffer, 1.25µL of 20X RT enzyme and 0.25µL of nuclease free water and incubated in a thermal cycler for 60 min at 37°C and 5 min at 95°C to inactivate the RT enzyme. Specific transcript preamplification was performed by adding 50µL of Taqman® PreAmp Master and 25µL of the Custom PreAmp Pool to the RT reaction mix (25µL) and incubated in a thermal cycler for 10 min at 95°C followed by 14 cycles at 95°C for 15 sec and 60°C for 4 min. The Custom PreAmp Pool contained 63 Taqman® assays. The same set of Taqman® assays were also printed onto the TLDA card and used for subsequent RT-PCR. The primers for RPS18 were not included in the custom pool due to the already high expression level of this gene. Note that each assay contained specific primers for transcript specific preamplification.
Preamplified cDNA was diluted to 1/32 by adding 12.5µL of cDNA from the completed RT reaction to 187.5µL of nuclease free water and 200µL of Taqman® Universal PCR Master Mix (no UNG). Two samples were loaded in each TLDA card (100 µL per port; 4 ports per sample). RT-PCR was performed using the ABI PRISM 7900HT.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA).
|
| Description |
Control
Preamplified cDNA from total RNA
|
| Data processing |
Amplification curves were processed using SDS 2.4 and analyzed using RQ Manager 1.2.1 to obtain mean cycle threshold (Ct) of the triplicate measurements for each gene.
The mean Ct for ACTB and RPS18 was used to calculate for the ∆Cts
|
| |
|
| Submission date |
Apr 05, 2018 |
| Last update date |
Apr 04, 2019 |
| Contact name |
Mark Magbanua |
| Organization name |
UCSF/Helen Diller Familiy Comprehensive Cancer Center
|
| Department |
HemOnc
|
| Lab |
Park
|
| Street address |
1450 3rd Street, PO Box 589001
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158-9001 |
| Country |
USA |
| |
|
| Platform ID |
GPL16020 |
| Series (2) |
| GSE112757 |
RNA profiling of EpCAM-positive cells in bone marrow of early breast cancer (EBC) patients |
| GSE112759 |
Parallel DNA and RNA profiling of EpCAM-positive cells in bone marrow and primary tumor tissue with positive disseminated tumor cell count via immunomagnetic Enrichment/Flow Cytometry of metastatic breast cancer patients confirm their malignant nature |
|
