Total RNA was reverse-transcribed using qScript XLT cDNA SuperMix (Quantabio) according to the manufacturer’s protocol. cDNA was used as a template for the real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on the 5′ nuclease chemistry with ABI PRISM 7500 sequence detection system (Applied Biosystems, Switzerland), using the following assay-on-demand reagents (Applied Biosystems): PARP1 (Hs 00242302_m1); TGFBR1 (Hs 00610318_m1); TGFBR2 (Hs 00234253_m1); BCL6 (Hs 00153368_m1). As previously reported,14 (link) PUM1 (Hs 00472881_m1) was used as a reference for normalisation and relative expression analyses. The comparative cycle threshold method (Applied Biosystems) was used for calculations of relative quantitation of targets.
Protocol detail
Quantitative RT-PCR for Gene Expression Analysis
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Assay
Bcl6
Cdna
Parp1
Prism
Real time polymerase chain reaction
Reverse transcriptase
Rt pcr
Tgfbr1
Tgfbr2
Corresponding Organization : University Hospital of Basel
Other organizations : Ruhr University Bochum
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Variable analysis
independent variables
- None explicitly mentioned
- Relative expression of PARP1, TGFBR1, TGFBR2, and BCL6 genes
- PUM1 gene expression for normalization and relative expression analyses
- Positive control: None mentioned
- Negative control: None mentioned
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