Quantifying Gene Expression in Cell Lines

NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C. HMECs and HMLER cells were cultured in HuMEC ready medium (12752010, Life Technologies). Dharmafect 2 reagent (Dharmacon) was used to transfect siRNA at 40 nM. The tetracycline-regulated rat Ubtf1 and Ubtf2 MEF-3T3 cell lines were established as reported in Sanij et al. (2008) (link). Cells were lysed, RNA was extracted, and first-strand cDNA was synthesized using random hexamer primers and Superscript III (Invitrogen). Quantitative PCR (qPCR) was performed in duplicate using the FAST SYBR Green dye on the StepOnePlus real-time PCR system (Applied Biosystems). Primer sequences are listed in (Supplemental Table 20).

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Sanij E., Diesch J., Lesmana A., Poortinga G., Hein N., Lidgerwood G., Cameron D.P., Ellul J., Goodall G.J., Wong L.H., Dhillon A.S., Hamdane N., Rothblum L.I., Pearson R.B., Haviv I., Moss T, & Hannan R.D. (2015). A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes. Genome Research, 25(2), 201-212.

Publication 2015

HuMEC ready medium Dharmafect 2 reagent Superscript III FAST SYBR Green dye StepOnePlus real-time PCR system

Cdna Cell lines Cells Cultured medium Eagle Fetal bovine serum Nih3t3 cells Primer Sirna Sybr green Tetracycline

Corresponding Organization :

Other organizations : Peter MacCallum Cancer Centre, University of Melbourne, University of Adelaide, Centre for Cancer Biology, University of South Australia, South Australia Pathology, Monash University, Université du Québec, Université Laval, University of Oklahoma, Bar-Ilan University, University of Queensland

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Variable analysis

independent variables

SiRNA transfection at 40 nM concentration using Dharmafect 2 reagent

dependent variables

MRNA expression levels measured by quantitative PCR (qPCR)

control variables

NIH3T3 cells cultured in DMEM with 10% FBS at 37°C
HMECs and HMLER cells cultured in HuMEC ready medium
Tetracycline-regulated rat Ubtf1 and Ubtf2 MEF-3T3 cell lines established as reported in Sanij et al. (2008)
Cell lysis, RNA extraction, and first-strand cDNA synthesis using random hexamer primers and Superscript III

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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