At 24, 48, 72, 96, 120, 144, and 168 h after transfection, RNA was isolated from pelleted cells using Qiagen RNEasy Mini Kits (Hilden, GER). RT-qPCR was performed using Luna Universal OneStep RT-qPCR kits (New England Biolabs, Ipswich, USA) on a Quantstudio 3 (Thermo Fischer, Waltham, USA) according to the manufacturer’s instructions. RT-qPCR primers for PBRM1 and GAPDH were obtained from Eurofins Genomics (Ebersberg, Germany; Sequence GAPDH-primer (Forward: 5′-CAA-TGA-CCC-CTT-CAT-TGA-CC-3′, and Reverse: 5′-TTG-ATT-TTG-GAG-GGA-TCT-GG-3′) and PBRM1-primer (Forward: 5′-AGG-AGG-AGA-CTT-TTC-AAT-CTT-CC-3′, and Reverse: 5′-CTT-CGC-TTT-GGT-GCC-CTA-ATG-3′). Relative gene expression was calculated by the ΔΔCT method44 (link),45 (link), using GAPDH expression levels to normalize PBRM1 expression levels.
Zimmer K., Kocher F., Untergasser G., Kircher B., Amann A., Baca Y., Xiu J., Korn W.M., Berger M.D., Lenz H.J., Puccini A., Fontana E., Shields A.F., Marshall J.L., Hall M., El-Deiry W.S., Hsiehchen D., Macarulla T., Tabernero J., Pichler R., Khushman M., Manne U., Lou E., Wolf D., Sokolova V., Schnaiter S., Zeimet A.G., Gulhati P., Widmann G, & Seeber A. (2023). PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system. NPJ Precision Oncology, 7, 64.
Other organizations :
Caris Life Sciences (United States), University Hospital of Bern, University of Bern, University of Southern California, Ospedale Policlinico San Martino, Sarah Cannon Research Institute, The Barbara Ann Karmanos Cancer Institute, Wayne State University, Georgetown University, Georgetown University Medical Center, Temple University Health System, Fox Chase Cancer Center, Brown University, Providence College, The University of Texas Southwestern Medical Center, Vall d'Hebron Hospital Universitari, Innsbruck Medical University, Universität Innsbruck, University of Alabama at Birmingham, University of Minnesota Medical Center, Ospedale di Bolzano, Rutgers, The State University of New Jersey
Time after transfection (24, 48, 72, 96, 120, 144, 168 h)
dependent variables
Relative gene expression of PBRM1
control variables
GAPDH expression levels used to normalize PBRM1 expression levels
controls
No positive or negative controls were explicitly mentioned in the input protocol.
Annotations
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