For qRT-PCR, total RNA (500 ng) was transcribed into cDNA using qScript™ cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD). Real-time RT-PCR reactions were performed using qScript™ One-Step SYBR® Green qRT-PCR kit (Quanta Biosciences, Gaithersburg, MD) and the following TMPRSS2-ERG fusion gene-specific primers: TMPRSS2-ERG RT forward, 5’-CAGGAGGCGGAGGCGGA-3’; TMPRSS2-ERG RT reverse, 5’-GGCGTTGTAGCTGGGGGTGAG-3’ and human GAPDH forward, 5’-GAGTCAACGGATTTGGTCGT-3’, and reverse, 5’-TTGATTTTGGAGGGATCTCG-3’ were used (25 (link)). qRT-PCR reactions were run on an ABI Prism 7000 Sequence Detection System (Thermoscientific, Grand Island, NY). Data were analyzed using the ΔΔCt method (26 (link)), where target gene expression is normalized to the housekeeping gene by taking the difference between Ct values for target gene and housekeeping gene (ΔCt). This value was then compared to that of the normalized control sample (ΔΔCt). The fold change was then determined by the formula: 2−ΔΔCt.
Protocol detail
Quantitative Real-Time PCR Analysis of TMPRSS2-ERG Fusion Gene
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qScript™ cDNA Synthesis Kit
qScript™ One-Step SYBR® Green qRT-PCR kit
ABI Prism 7000 Sequence Detection System
Cdna
Gapdh
Gene
Gene expression
Housekeeping gene
Human
Primers
Prism
Rt pcr
Sybr green
Synthesis
Tmprss2
Corresponding Organization : University of Miami
Other organizations : Thomas Jefferson University
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Variable analysis
independent variables
- None explicitly mentioned
- Expression of TMPRSS2-ERG fusion gene
- 500 ng of total RNA used for cDNA synthesis
- GAPDH used as a housekeeping gene for normalization
- Positive control: None mentioned
- Negative control: None mentioned
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