Total RNA was isolated from benign prostate and tumor tissues, as well as from prostate cancer cells, using the Qiagen RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Complementary DNA was produced by PrimeScript (Takara, Kyoto, Japan). qPCR was performed in a StepOne PCR System (Thermo Fisher) using KAPA SYBR Green (Sigma). Gene expression was calculated by ΔΔCt method and normalized to GAPDH at the same condition16 (link). Details of the PCR primers are listed in Supplementary Table 3 .
Kapa sybr green
Manufactured by Merck Group
KAPA SYBR Green is a fluorescent dye used in quantitative real-time PCR (qPCR) experiments. It binds to double-stranded DNA and emits a fluorescent signal that can be detected and monitored during the PCR amplification process. The dye is used to quantify target DNA sequences in a sample.
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Lab products found in correlation
3 protocols using kapa sybr green
1
Prostate Cancer RNA Quantification Protocol
2
RNA Isolation and Real-Time PCR Analysis
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RNA was isolated using the ISOGEN II (Nippon Gene, Tokyo Japan) in accordance with manufacturer’s instructions. Complementary DNA was synthesized from equivalent concentrations of total RNA using Prime Script (TAKARA bio, Kyoto Japan) in accordance with manufacture’s protocols. Amplification was performed in a StepOne PCR System (Thermo Fisher, Waltham, MA) using KAPA SYBR Green (Sigma). Fold changes for experimental groups relative to the loading control were calculated by ΔΔCt method. Primer sequences were provided in the Supplementary Table 2 .
3
Quantitative gene expression analysis
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RNA was isolated using the ISOGEN II (Nippon Gene) in accordance with manufacturer's instructions. Complementary DNA was synthesized from equivalent concentrations of total RNA using Prime Script (TAKARA bio) according to manufacturer's protocols. Amplification was performed in a StepOne PCR System (Thermo Fisher Scientific) using KAPA SYBR Green (Sigma). Fold changes for experimental groups relative to the loading control (GAPDH, MB) were calculated by DDC t method. Primer sequences were described before (15, 18, 27) and Supplementary Table S1.
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