Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - pilot dataset (qPCR validation) - Dataset (ID:20325)
|HMS Dataset ID:||20325|
|Dataset Title:||Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - pilot dataset (qPCR validation)|
|Screening Lab Investigator:||Huan (Sharon) Wang, Sarah Boswell|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||To define molecular network markers of tyrosine kinase inhibitor-induced cardiotoxicity, we treated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with one of four tyrosine kinase inhibitors displaying a range of mild to severe effects on heart function (Erlotinib, Lapatinib, Sorafenib, or Sunitinib) or vehicle control (DMSO). We performed 3 different treatments for each drug, including two doses (3.16 and 10 µM) at 24 hours and the lower dose (3.16 µM) at 72h. Gene expression changes were assessed at the cell population level using qPCR. This is a validation dataset for RNA-seq HMS-LINCS Dataset #20324.|
1. Cryo-preserved Cor.4U hiPSC-CMs (Ncardia, previously Axiogenesis) were thawed into 75cm2 flasks for two days before reseeding into multi-well plates in order to remove cells that died during the thawing process. |
2. Media was changed every other day during culture.
3. Cor.4U hiPSC-CMs were cultured in BMCC media (Ncardia, previously Axiogenesis) containing 1% fetal bovine serum during drug treatment.
4. The same batch of Cor.4U cells was used for each of the three technical replicates of drug treatment conditions in this experiment.
5. Cells were washed once with PBS with no calcium or magnesium, scraped in RLT buffer (RNeasy Mini Kit, Qiagen), and cell lysates were frozen at -80 °C until further processing.
6. At the time of RNA purification, samples were thawed at room temperature and processed with QIAshredder (Qiagen) to ensure complete lysis.
7. Total RNA purification was performed following manufacturer’s instructions, including a 30 minute on-column DNase digestion step. Sample concentrations were determined by Nanodrop and RNA quality was assessed on a subset of samples by Bioanalyzer (Agilent); all samples scored RINs of > 9.0.
8. Samples were reverse transcribed to cDNA using an RT2 First Strand Kit (Qiagen, Cat. No. 330404).
9. Gene expression of 90 selected genes and four housekeeping genes (ACTB, GUSB, NONO, RPLP0) were measured based on custom RT2 profiler PCA arrays using RT² SYBR Green ROX qPCR Mastermix (Qiagen, Cat. No. 330524) and Applied Biosystems QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific). Experiments were performed by the Qiagen Lifesciences Service Core (Frederick, MD). .
10. Ct values were exported to a text file and analyzed using R, and normalized to the average Ct values of three of the housekeeping genes (GUSB, NONO, RPLP0) whose expression was determined to be unchanged among the experimental conditions.
|HMS Dataset Type:||qPCR|
|Date Publicly Available:||2018-03-20|
|Most Recent Update:||2018-03-23|