RNA isolation and real-time PCR were performed as described (Pedraza et al. 2008 (link)). For RNA isolation, cells were seeded onto PDL/Fibronectin/Laminin-coated 10 cm dishes. After 5 days treatment, cells were washed twice with cold PBS, 1 ml of Trizol reagent (Invitrogen) was added, and the plate was incubated on ice for 10 min. The cells in the Trizol reagent were then collected and RNA was extracted in phenol/chloroform. cDNA was generated from 1 μg total RNA by Superscript II (Invitrogen) reaction and transcripts were detected and amplified by qRT-PCR in a Light Cycler machine (Roche, Indianapolis, IN), following the manufacturer's instructions.
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RNA Extraction and qRT-PCR Analysis
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Corresponding Organization : University of Colorado Denver
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Variable analysis
independent variables
- Treatment duration (5 days)
- MRNA expression levels (detected and amplified by qRT-PCR)
- Cell seeding onto PDL/Fibronectin/Laminin-coated 10 cm dishes
- Washing cells with cold PBS prior to Trizol reagent addition
- RNA extraction in phenol/chloroform
- CDNA generation from 1 μg total RNA by Superscript II reaction
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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