Abi real time fluorescence quantitative pcr platform

Total RNA in tissue samples and cells was harvested using Trizol reagent (Takara Holdings Inc., Kyoto, Japan). Taqman Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Reverse Transcript Kit (Applied Biosystems, Inc., Foster City, CA, USA) were then utilized for reverse transcription. Subsequently, the expression of ZFPM2-AS1 was detected by SYBR Green quantitative PCR Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) kit with ABI real-time fluorescence quantitative PCR platform (Thermo Fisher). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were utilized as housekeeping genes. Details of primers are listed in Table 2.

Trizol regent (Takara Bio Inc., Otsu, Shiga, Japan) was used for total RNA extraction from cultured cells and tissues. RNA was reversely transcribed to cDNA using PrimeScript RT Master Mix (Takara). Subsequently, real-time PCR was performed using SYBR Green qPCR Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) on an ABI real-time fluorescence quantitative PCR platform (Thermo Fisher). All primers were purchased from Invitrogen (Shanghai, China). The data were processed using the 2^−ΔΔCt relative expression method. To reduce errors, the experiment was repeated three times and Ct was averaged. miR-579 was normalized to U6, and lncRNA HCG11 and MMP13 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences are listed below: miR-579 forward: 5ʹ-CGTGCCGTTCATTTGGTATAAAC-3ʹ and reverse: 5ʹ-GAGCAGGGTCCGAGGT-3ʹ; LncRNA HCG11 forward: 5ʹ-GCTCTATGCCATCCTGCTT-3ʹ and reverse: 5ʹ-TCCCATCTCCATCAACCC-3ʹ; MMP13 forward: 5ʹ-GACTTCCCAGGAATTGGTGA-3ʹ and reverse: 5ʹ-TGACGCGAACAATACGGTTA-3ʹ; GAPDH forward: 5ʹ-GAGTCCACTGGCGTCTTCAC-3ʹ and reverse: 5ʹ-ATCTTGAGGCTGTTGTCATACTTCT-3ʹ; U6 forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ.