Samples from 4-month-old mice were snap-frozen in liquid nitrogen and stored at −80°C. Total RNAs were isolated using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s protocol. Purity of RNAs was assessed by a ratio of absorbance at 260 and 230 nm > 1.7. RNA quality was checked on agarose gel. One microgram of RNA was used for reverse transcription with the Maxima First Strand complementary DNA (cDNA) Synthesis Kit for reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Fermentas). cDNAs were amplified using the Maxima SYBR Green qPCR Master Mix (2×; Fermentas). qPCR reactions were performed on a Roche Light Cycler system (Roche). All PCR and qPCR products were examined qualitatively on agarose gels. All presented RT-qPCR results were normalized by the geometrical mean of three independent reference genes (Ywhaz, Polr2a, and Rplpo), as previously described (49 (link)). Sequences of primers are listed in table S6.
Protocol detail
Quantifying gene expression in mice
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Corresponding Organization :
Other organizations : Institut Mondor de Recherche Biomédicale, Inserm, Université Paris-Est Créteil, Université Paris-Saclay, Université Paris-Sud, Laboratory Signaling and Cardiovascular Pathophysiology, Unit of Functional and Adaptive Biology, Université Paris Cité, Centre National de la Recherche Scientifique, Université Bourgogne Franche-Comté, Institut Agro Dijon, Université de Bordeaux, Microbiologie de l’alimentation au service de la santé, AgroParisTech, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Université d'Évry Val-d'Essonne, HIA du Val-de-Grâce à Paris, Laboratoire Jean Perrin, Sorbonne Université
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Variable analysis
independent variables
- None explicitly mentioned
- Gene expression levels measured by RT-qPCR
- Age of mice (4-month-old)
- Tissue collection method (snap-frozen in liquid nitrogen)
- Storage temperature (-80°C)
- RNA extraction method (TRIzol reagent)
- RNA purity assessment (260/230 nm ratio > 1.7)
- RNA quality check (agarose gel)
- Reverse transcription method (Maxima First Strand cDNA Synthesis Kit)
- QPCR amplification method (Maxima SYBR Green qPCR Master Mix)
- QPCR platform (Roche Light Cycler system)
- Normalization of RT-qPCR results (geometric mean of three reference genes: Ywhaz, Polr2a, and Rplpo)
- None explicitly mentioned
- None explicitly mentioned
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