Cells were nonenzymatically isolated from 3D-culture (50,000 cells/well; 500 μL Cultrex/well in 12-well plate) using the Cultrex 3D-Culture Cell Harvesting Kit (Trevigen), and total RNA was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. One μg of total RNA was subsequently reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) and subjected to quantitative real-time PCR using 1× iQ SYBR Green Supermix (Bio-Rad), as previously described (118 (link)) and using the primers listed in table S2 . Changes in gene expression were determined using the ΔΔCt method.
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Isolating and Quantifying RNA from 3D-Cultured Cells
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Other organizations : Case Western Reserve University, University School, Cleveland Clinic
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Variable analysis
independent variables
- 3D-culture (50,000 cells/well; 500 μL Cultrex/well in 12-well plate)
- Total RNA extracted
- Gene expression changes determined using the ΔΔCt method
- Nonenzymatic isolation of cells using the Cultrex 3D-Culture Cell Harvesting Kit (Trevigen)
- RNA extraction using the RNeasy Mini Kit (QIAGEN)
- Reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad)
- Quantitative real-time PCR using 1× iQ SYBR Green Supermix (Bio-Rad)
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