Quantitative Analysis of miR-29a-3p Expression

miR-29a-3p Oligod (T) specific RT primers were designed. Cells were dissolved in 1 mL Trizol (Thermo Fisher Scientific, Waltham, MA, USA) to extract the total RNA based on instructions. RNA samples in which the miR-29a-3p expression was detected were treated with 3’ poly-A tail. RNAs were reversed transcript into cDNA using M-MLV reverse transcriptase (D7160L, Beyotime, Shanghai, China) and random primers. The PCR reaction system and reaction conditions were prepared based on the instructions of Premix Ex Taq™II kit (Takara, Dalian, China). ABI7500 quantitative PCR apparatus (Applied Biosystems, Shanghai, China) was used for RT-PCR with U6 as internal control of miRNA and GAPDH of mRNA. Data were analyzed using 2^-ΔΔCt method ^{22 (link)}: ΔΔCt = [Ct_{(target gene)}-Ct_{(internal gene)}]_{experimental group} -[Ct_{(target gene)}-Ct_{(internal gene)}]_{control group.} The primers for gene are listed in Table 1.