Topscript reverse transcriptase kit

The real-time PCR primers for the RPL21 gene (primers 15 and 16; Table 1) were designed according to the mRNA sequences cloned in this study. The cDNAs were synthesized from total RNA that was extracted from 7 different tissues (kidney, heart, liver, lung, ovary, intestine, and skin) and 3 types of cell lines (wild-type fibroblast, transient RPL21-overexpressed fibroblast, and RPL21-knockdown fibroblast) by using the TOPscript Reverse Transcriptase kit (Enzynomics, Korea). The RPL21 expression levels of the different pig tissues were compared by using a standard curve method as previously described [19 (link)]. Briefly, a standard curve was constructed with serial ten-fold dilutions of the pCX-RPL21 plasmid DNA by performing real-time PCR. Then the cycle threshold (Ct) value that was obtained from each sample was transformed into a copy number according to the standard curve. The PCR was performed with primers 15 and 16 by using the TOPreal One-step RT qPCR Kit (Enzynomics) with the following conditions: 40 cycles of 10 sec denaturation at 95℃, 15 sec annealing at 64℃ with a 0.1℃ decrease in each cycle for 30 cycles, and a 20 sec extension at 72℃. When comparing the RPL21 expression levels in the same tissue or cell line, we normalized the expression level against a housekeeping gene, GAPDH (primers 13 and 14).