Following the established methods (Li et al., 2002), reserve mesenchyme was rapidly separated from five morphologically defined layers (dermis, reserve mesenchyme, precartilage
, transition zone and cartilage; Fig.
Expression analysis by real-time quantitative RT-PCR reveals that CaM gene has a higher expression level in antler skin layer than in reserve mesenchyme layer, precartilage layer and cartilage layer, indicating that this gene may play a regulatory role in rapid growth of antler skin.
Different tissue layers (skin, reserve mesenchyme, precartilage, cartilage) of the growing antlers were determined as described by Li et al.
Total RNAs using SV Total RNA Isolation System reagen (Promega) were isolated from different tissues including skin, mesenchyme, precartilage and Cartilage.
In a real-time RT-PCR study, specific primers (CaM-F: 5'-GGTCAGAACCCAACAGAAG-3' and CaM-R: 3'-CTTCACTGTCGGTGTCTTTC-5') were used to amplify a 130 bp fragment with cDNA from skin, reserve mesenchyme, precartilage and cartilage, organs using 18S as a positive control.
A multiple comparisons (Duncan's) test was conducted to compare significant differences in CaM expression between skin, mesenchyme, precartilage and cartilage, using the SPSS13.0 software.