Quantitative real time pcr machine

Normal AF-MSCs and ICHD AF-MSCs were cultured in CMEM with 10 µM 5′-azacytidine (5′-aza; Sigma-Aldrich MO, USA) to induce cardiomyogenic lineages. After 24 h, the medium was replaced with CMEM without 5′-aza for 21 days, with the medium changing twice a week. Control cells were treated with CMEM alone. The cells were examined for fold changes in the expression of cardiac transcription factors by real-time PCR. RNA was isolated from untreated and 5′-aza-treated normal and ICHD AF-MSCs using the Trizol reagent (Invitrogen, Waltham, MA, USA). A high-capacity cDNA reverse transcription kit was used to transcribe the mRNA to cDNA (Bio-Rad). SYBR Green PCR Master Mix Chemistry was used to analyze the expression of cardiac transcription factors viz. GATA-4, ISL-1, NKx 2-5, TBX-5, TBX-18, and MeF-2C using a quantitative real-time PCR machine (Bio-Rad). The ∆∆Ct method was used to determine the relative fold change expression level for each targeted gene normalized with a housekeeping gene, i.e., GAPDH, and fold change expression in the genes was calculated by the 2^−∆∆Ct method [21 (link)]. The primers used in quantitative real-time PCR are given in Table 2.

Total RNA was isolated from untreated and tri-lineage differentiated normal and ICHD AF-MSCs using the Trizol reagent (Invitrogen, Waltham, MA, USA). A high-capacity cDNA reverse transcription kit was used to transcribe the mRNA to cDNA (Bio-Rad, Hercules, CA, USA). SYBR Green PCR Master Mix Chemistry was used to analyze the expression of adipogenic gene viz. PPARγ, lipoprotein lipase, osteogenic genes viz. RUNX and Osteopontin, chondrogenic gene viz. SOX9 and AGCAN using a quantitative real-time PCR machine (Bio-Rad). The ∆∆Ct method was used to determine the relative fold change expression level for each targeted gene normalized with a housekeeping gene, i.e., GAPDH, and fold change expression in the genes was calculated by the 2^−∆∆Ct method [21 (link)]. The primers used in quantitative real-time PCR are given below in Table 2.

Total RNA was isolated from both normal and ICHD AF-MSCs using the Trizol reagent (Invitrogen, Waltham, MA, USA). A high-capacity cDNA reverse transcription kit was used to transcribe the mRNA to cDNA (Bio-Rad). SYBR Green PCR Master Mix Chemistry was used to analyze the expression of senescence-associated genes viz. TP53 and CDKN1A and DNA damage-response genes viz. MRE11, NBS1, and PARP, using a quantitative real-time PCR machine (Bio-Rad). The ∆∆Ct method was used to determine the relative fold change expression level for each targeted gene normalized with a housekeeping gene, i.e., GAPDH, and fold change expression in the genes was calculated by the 2^−∆∆Ct method [21 (link)]. The primers used in quantitative real-time PCR are given above in Table 2.