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Quantification of mRNAs by real-time RT-PCR

CB Christian Boyer
LC Laura Cussonneau
CB Charlotte Brun
CD Christiane Deval
JB Jean-Paul Pais de Barros
SC Stéphanie Chanon
NB Nathalie Bernoud-Hubac
PD Patricia Daira
AE Alina L. Evans
JA Jon M. Arnemo
JS Jon E. Swenson
GG Guillemette Gauquelin-Koch
CS Chantal Simon
SB Stéphane Blanc
LC Lydie Combaret
FB Fabrice Bertile
EL Etienne Lefai
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For mRNA quantification using RT-qPCR, total RNAs were obtained from muscle and adipose tissues collected at the two time points (S and W). For the muscle tissue, RNAs were extracted from 8 bears in summer and winter, while for adipose tissue, RNAs were extracted from 5 bears in summer and 13 bears in winter (Supplementary Table S1).

Muscle and adipose tissue total RNA was isolated using the TRIzol reagent (Invitrogen, Courtaboeuf, France) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from 1 μg of total RNA using the PrimeScript RT kit (Ozyme, saint quentin en Yveline, France) with a mixture of random hexamers and oligo(dT) primers, and treated with 60 units of RnaseH (Ozyme). Real-time PCR assays were performed with Rotor-Gene 6000 (Qiagen, Courtaboeuf, France). The primers and real-time PCR assay conditions are listed in supplementary Table S4. The results were normalized by using TBP (TATA box binding protein) mRNA concentration, measured as reference gene in each sample.

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