Igf1 mm00439560 m1

Total RNA was isolated and extracted from cell lysates using TRI-Reagent
(Sigma–Aldrich). The quality and quantity of RNA recovered were assessed
by running on 1.5% agarose gel and Nanodrop spectrophotometer. RT-PCR used
1 μg RNA per sample and was carried out using the reverse
transcription kit (CODE) Applied Biosciences (kit code is 4368814). Specific
mRNA levels were determined using an ABI 7900 sequence detection system (Applied
Biosystems). Reactions were carried out in 12.5 μl volumes on 384 well
plates (Applied Biosystems) in a reaction buffer containing 2× Taqman
Universal PCR Master Mix (Applied Biosystems). Primers and probes for specific
genes were purchased in ‘Assay on Demand’ format from Applied
Biosystems (IGF1:Mm00439560_m1, MYOD:Mm00521984_m1,TP53:Mm00480750_m1,
H6PD:Mm00557617). These were normalised against 18S rRNA (Applied Biosystems) as
an internal control. Raw data were recovered as CT values and
analysed as per the 2^−ΔΔCTmethod.
RT-PCR of alternative transcripts from the Hsd11b1 gene was
carried out as described in Staab et
al. (2011).

RNA was extracted from tissues using TRIzol (MDBio Inc., Taipei, Taiwan). Synthesis of cDNA was achieved using MMLV RTase (Promega, Madison, Wisconsin, USA). Synthesized cDNA was used as the template for semi-quantitative RT-PCR and real-time quantitative PCR. The primer sequences were as follows: mouse growth hormone (GH): forward, 5′-CAGCCTGATGTTCGGCACCTCGGA-3′ and reverse, 5′-GCGGCGACACTTCATGACCCGCA-3′; mouse IGF-1: forward, 5′-CTGGACCAGAGACCCTTTGC-3′ and reverse, 5′-AGAGCGGGCTGCTTTTGTAG-3′; mouse GAPDH: forward, 5′-GCCATCAACGCCCCTTCATT-3′ and reverse, 5′-ACGGAAGGCCATGCCAGTGAGCTT-3′. Mouse GH (Mm00433590_g1); IGF-1 (Mm00439560_m1); and GAPDH (Mm99999915_g1) TaqMan probes were purchased from Applied Biosystems (USA). The data were analyzed by the StepOne Real-Time PCR system (ABI, USA). The mRNA levels of GH and IGF-1 were normalized to that of GAPDH and expressed relative to the control using the formula 2^-ΔΔCT.