Quantifying mRNA Levels by Real-Time qPCR

To quantify mRNA-levels real-time qPCR was performed using Maxima-SYBR-Green-qPCR-Master-Mix (Thermofisher, Waltham, USA) and GAPDH as reference-gene as previously described (Hagenbuchner et al., 2017 (link)): total-RNA was isolated from 5 × 10⁶ cells using TRI-Reagent (Merck, Vienna, Austria) and 1 µg was reverse-transcribed to cDNA using RevertAid-H-Minus-cDNA-Synthesis Kit (Thermo Scientific, Waltham, USA). Oligonucleotides for BIM, NOXA, DEPP1, SESN3, and GAPDH are listed in Supplementary file 2. qRT-PCR-reactions were performed in triplicates in a Bio-Rad-iCycler-instrument and repeated 3-times. After normalization to GAPDH, regulation was calculated between treated and untreated cells.

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Hagenbuchner J., Obsilova V., Kaserer T., Kaiser N., Rass B., Psenakova K., Docekal V., Alblova M., Kohoutova K., Schuster D., Aneichyk T., Vesely J., Obexer P., Obsil T, & Ausserlechner M.J. (2019). Modulating FOXO3 transcriptional activity by small, DBD-binding molecules. eLife, 8, e48876.

Publication 2019

Maxima-SYBR-Green-qPCR-Master-Mix TRI-Reagent RevertAid-H-Minus-cDNA-Synthesis Kit iCycler-instrument

Cdna Cells Gapdh Gene Mrna Oligonucleotides Sybr green Synthesis

Corresponding Organization :

Other organizations : Innsbruck Medical University, Czech Academy of Sciences, Czech Academy of Sciences, Institute of Physiology, Tyrolean Cancer Research Institute, Universität Innsbruck, Charles University, Paracelsus Medical University

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Variable analysis

independent variables

Treatment (treated vs. untreated cells)

dependent variables

MRNA levels of BIM, NOXA, DEPP1, and SESN3

control variables

Cell number (5 × 10^6 cells)
Reference gene (GAPDH)
Reverse transcription kit (RevertAid-H-Minus-cDNA-Synthesis Kit)
QPCR master mix (Maxima-SYBR-Green-qPCR-Master-Mix)
QPCR instrument (Bio-Rad-iCycler)

controls

Positive control: Not explicitly mentioned
Negative control: Untreated cells

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