RNA was extracted from VCaP and LNCaP cells using the Trizol (Invitrogen, Carlsbad, CA,) method. For RT-PCR experiments, cDNA was amplified using an Eppendorf Mastercycler Realplex2 qPCR machine. PCR products were loaded onto a 0.8% agarose gel containing ethidium bromide. For RT-qPCR experiments, cDNA was amplified with SYBR green from the SensiFAST SYBR kit obtained from Bioline (Taunton, MA, BIO-98002). Gene expression was quantified with previously described qPCR method (33 (link)). Fold changes in message levels were quantified using comparative Ct method (34 (link)) . PCR primers are listed in Supplementary Table 2 .
Mastercycler realplex2 qpcr machine
Manufactured by Eppendorf
The Mastercycler Realplex2 is a real-time qPCR machine designed for quantitative gene expression analysis. The device is capable of performing thermal cycling and real-time detection of fluorescent signals during the PCR amplification process.
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2 protocols using mastercycler realplex2 qpcr machine
1
RNA Extraction and qPCR Analysis in VCaP and LNCaP Cells
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using the E.N.Z.A. Micro-elute Total RNA Kit I as per the manufacturer's protocol (Omega Bio-tek) and reverse transcribed using a Quantitect Reverse Transcription Kit (QIAGEN). For qPCR analysis, a cDNA template was amplified with the KAPA SYBR FAST Universal qPCR Master Mix (Kapa Biosystems) as per the manufacturer's protocol using the primers listed in the Supplemental Information . Reactions performed in duplicate were performed using a Mastercycler Realplex2 qPCR machine (Eppendorf, Mississauga, ON). Data were analyzed using the delta-delta CT method to quantify gene expression (Schefe et al., 2006 (link)) and expressed as means ± SEM of three independent experiments.
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