Sybr green reaction mix

Total RNA was isolated and purified from 30–50 mg of TA muscle using Trizol Reagent (Invitrogen), according to the manufacturer’s protocol. One microgram of total RNA was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was performed with the SDS-ABI Prism 7500 (Applied Biosystem, Life Technologies, Monza, IT), using the Sybr Green reaction mix (Applied Biosystem).
The following primer sequences were used:
atrogin1 for: GCA AAC ACT GCC ACA TTC TCT C
atrogin1 rev: CTT GAG GGG AAA GTG AGA CG
MuRF-1 for: ACC TGC TGG TGG AAA ACA TC
MuRF-1 rev: CTT CGT GTT CCT TGC ACA TC
myostatin for : TGC TGT AAC CTT CCC AGG ACC A
myostatin rev: GTG AGG GGG TAG CGA CAG CAC
GAPDH for ACC CAG AAG ACT GTG GAT GG
GAPDH rev CAC ATT GGG GGT AGG AAC AC
Five control healthy mice, six C26-bearing mice, six C26 WR, six vehicles, and five mice treated with either AICAR or rapamycin were analyzed.

The protocol for real-time PCR has been described before [27] (link). Briefly, total RNA was extracted from the C2C12 and Sol8 myoblasts using TRIZOLE (Life Technology; Rockville, MD) according to the supplier’s instruction. Then, the first strand of cDNA was synthesized using the Superscript II kit (Life Technology; Rockville, MD). Real time PCR was performed in a 25 µl reaction mixture containing 5 µM forward/reverse primers, 1X SYBR Green reaction mix (Applied Biosystem; Werrington, UK), and various amounts of template. Different amounts of template were used in the same reaction to make sure the linear amplification of PCR products. Gapdh was used as internal control amplified in the same PCR assay. The primer sets used for quantification of myogenic gene expression are listed as in table 1. All reactions were performed in ABI 7300 sequence detection system.

RNA was extracted from 250 mg of skin tissue using TRIzol® protocol (Invitrogen®). One hundred nanograms were used for real-time PCR analysis. PCR was performed in a 15 µL reaction mixture containing 7.5 µL 2x SYBR Green Reaction Mix (Invitrogen), 0.3 µL each primer (10 pmol), 0.3 µL SuperScript III RT/Platinum Taq Mix (10pmol/µL), 0.15 µL ROX Reference Dye, and 5 µL sample in water. Gene specific primers were used. β2M : Sense, ACC CAC ACT GTG CCC ATC TA, Antisense, GCCACAGGATTCCATACCCA; TGF-β : Sense, GGA GAC GGA ATA CAG GGC TTT C, Antisense, CGG TTC ATG TCA TGG ATG GTC; Collagen I : Sense, AAA CTT TGC TTC CCA GAT GT C; Antisense, GGA CCC ATT GGA CCT GAA C; Collagen III : Sense, CCC ATG ACT GTC CCA CGT AAG C; Antisense, CCA GCT GCA CAT CAA CGA CAT C. The result was calculated under the 2^−ΔΔCT ratio of the housekeeping gene expression β2M (β2 microglobulin). Reactions were performed using StepOne™ Real-Time PCR System (Applied Biosystem).

One hundred nanograms were used for real-time PCR analysis. PCR was performed in a 15 μL reaction mixture containing 7.5 μL 2 × SYBR Green Reaction Mix (Invitrogen Life Technologies, Carlsbad, CA, USA), 0.3 μL each primer (10 pmol), 0.3 μL Super Script III RT/Platinum Taq Mix (10 pmol/μL), 0.15 μL ROX Reference Dye and 5 μL sample in water. Gene-specific primers were used. Reactions were performed using a StepOne™ Real-Time PCR System (Applied Biosystem, Foster City, CA, USA).