Cells were starved with phenol red-free and serum free-medium for at least 48 h and treated with the indicated drugs and/or androgens. RNA was extracted with a GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich). cDNA was synthesized from 1 μg RNA in a reverse transcription reaction using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Quantitative PCR (qPCR) analysis was conducted in triplicate with primers for PSA, TMPRSS2, FKBP5, and RPLPO described previously3 (link), with an ABI 7500 Real-Time PCR machine (Applied Biosystems) using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad) in 96-well plates at a final reaction volume of 20 μL. Accurate quantitation of each mRNA was achieved by normalizing the sample values to RPLPO and to vehicle-treated cells.
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Quantitative PCR Analysis of Prostate Cancer Markers
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GenElute Mammalian Total RNA miniprep kit
iScript cDNA Synthesis Kit
ABI 7500 Real-Time PCR machine
iTaq Fast SYBR Green Supermix with ROX
Androgens
Cdna
Cells
Drugs
Fast green
Fkbp5
Mammalian
Mrna
Primers
Real time pcr
Reverse transcription
Serum
Synthesis
Tmprss2
Corresponding Organization :
Other organizations : Cleveland Clinic Lerner College of Medicine, Cleveland Clinic, University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Drugs
- Androgens
- MRNA expression of PSA
- MRNA expression of TMPRSS2
- MRNA expression of FKBP5
- Phenol red-free and serum-free medium for at least 48 h
- RPLPO mRNA expression (used for normalization)
- Vehicle-treated cells (negative control)
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