MIR-423-3P USED AS REFERENCE GENE FOR MIRNA 146 A IN CELL LINES HEP-G2

Objective : We explored the stable reference genes for miRNA 146 A by RT-PCR in the cell lines Hep-G2 that were treated chlorogenic acid. We must do a series of tests in order to get a reference genes. Based on the literature there are 7 candidates recommended reference genes; mir-423-3p, 423-5p, 191, 103, 21, 16, and let-7a. We conducted using four candidates reference gene; mir-423-3p, 103, 16, and 21. Method s : In vitro study was performed in Hep-G2 cells. The samples were divided into control group and experiment group treated with 727μM chlorogenic acid. Samples were analyzed at 0, 2, 8 , 18, and 24 hours after being treated with chlorogenic acid. Total RNA was isolated from Hep-G2 with RNA extraction kit (miRCURYTM RNA Isolation Kit-Cell and plant Exiqon, Code Number 300110) and reverse transcribed to cDNA with Primerscript RT Reagent Kit (miRCURY LNATM Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit Exiqon, Code Number 203300). The primers for miRNA 146 A were Code Number 204688 from Exiqon (forward and reverse), and quantitative real time reverse transcription polymerase chain reaction was performed using SYBR Master Mix with Code Number 203450 (Exicon). MiRNA profiling was performed in four pairs of mir, consist of; mir-423-3p, 103, 21, and mir-16. By using the mean expression value of all expressed mir, we identified the most stable candidate reference genes for subsequent validation with normfinder software. Results: Data from RT-PCR were analyzed using Normfinder software. We found out that mir expression with good stablity is mir-103 and the best combination from two genes are mir-103 and mir-423-3p. Finally mir-423-3p is found more stable than mir-16, 21, and 103. Conclusion: We conclude the stable reference genes for miRNA 146 A treated with chlorogenic acid is mir-423-3p.