Retinoic Acid Induced Transcriptome

hESCs were exposed to all-trans retinoic acid (ATRA in DMSO; MP Biomedicals 02190269.6) at a final concentration of 1 µM in plain TESR-E8 medium supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific 15140122). Medium was changed daily. Cells were harvested in mirVana miRNA isolation kit lysis buffer (Ambion AM1560) after 0 d (no ATRA) or 1, 3, and 5 d following ATRA induction. Real-time qPCR was used to quantify levels of mRNA expression of four selected genes as previously described in Gingold et al. (2014) (link) and Sagi et al. (2016) (link). First strand cDNA was synthesized from 100 ng of total RNA using SuperScript III RT (Thermo Fisher Scientific 18080093) and random hexamers according to the manufacturer's instructions. qPCR was performed using SYBR Green PCR master mix (Applied Biosystems 10187094) on an Applied Biosystems 7500 real-time PCR system (primer sequences are listed in Supplemental Table S1).

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Scheepbouwer C., Aparicio-Puerta E., Gomez-Martin C., Verschueren H., van Eijndhoven M., Wedekind L.E., Giannoukakos S., Hijmering N., Gasparotto L., van der Galien H.T., van Rijn R.S., Aronica E., Kibbelaar R., Heine V.M., Wesseling P., Noske D.P., Vandertop W.P., de Jong D., Pegtel D.M., Hackenberg M., Wurdinger T., Gerber A, & Koppers-Lalic D. (2023). ALL-tRNAseq enables robust tRNA profiling in tissue samples. Genes & Development, 37(5-6), 243-257.

Publication 2023

penicillin/streptomycin mirVana miRNA isolation kit lysis buffer SuperScript III RT SYBR Green PCR master mix 7500 real-time PCR system

Atra Buffer Cdna Cells Dmso Expression genes Hescs Isolation Mirna Mrna Penicillin Primer Streptomycin Sybr green

Corresponding Organization :

Other organizations : Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, Universidad de Granada, Medisch Centrum Leeuwarden, Amsterdam Neuroscience, University of Amsterdam, University Medical Center Utrecht, Princess Máxima Center

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Variable analysis

independent variables

All-trans retinoic acid (ATRA) in DMSO

dependent variables

MRNA expression levels of four selected genes

control variables

Plain TESR-E8 medium
1% penicillin/streptomycin
Medium changed daily

controls

No ATRA exposure (0 days)

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