CXCL12 injection into the peritoneal cavities and RT-PCR

We injected 15 μg of CTX into a right TA muscle in six 10-week-old male C57BL/6 mice. CXCL12 was injected into the peritoneal cavities of three of these mice on day 2. We killed these mice on day 6 by using cervical dislocation and removed the TA muscles by microdissection. We homogenized these muscles and isolated RNA from these samples by using the RNeasy Mini Kit (QIAGEN). We achieved total RNA purification and first-strand cDNA synthesis. Amplification of PAX7, MyoD, and GAPDH by PCR was performed by using Ampdirect Plus (Shimazu, Kyoto, Japan) and NovaTaq DNA Polymerase (Novagen, Darmstadt, Germany) according to the manufacturers’ instructions. Primer sequences were as follows: PAX7 forward primer: 5′-CCGTGTTTCTCATGGTTGTG-3′, reverse primer: 5′-GAGCACTCGGCTAATCGAAAC-3′. MyoD forward primer: 5′-AGCACTACAGTGGCGACTCA-3′, reverse primer: 5′-GCTCCACTATGCTGGACAGG-3′. GAPDH forward primer: 5′-TGATGACATCAAGAAGGTGGTGAAG-3′, reverse primer: 5′-TCCTTGGAGGCCATGTAGGCCAT-3′.