Sybr green 1 real time pcr kit

Total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized by using TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The qRT-PCR assay was performed using a SYBR Green I Real-Time PCR Kit (GenePharma, Shanghai, China) to detect the gene expression. The following thermocycling conditions were used for the PCR: Initial denaturation at 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 20 s; and a final extension at 72 °C for 10 min. After amplification, a melting curve was generated to evaluate the specificity of PCR products at the end of each PCR cycle. And the amplification of only one product in qRT-PCR was confirmed by a melting curve analysis. U6 was used as an internal control, and the relative expression of miR-142-5p was calculated using the comparative delta CT (2^−ΔΔCt) method. The primers used were as follows: miR-142-5p forward, 5′- GGGCAUAAAGUAGAAAGC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

Total RNA, inclusive of the small RNA fraction, was extracted from cultured cells with TRIzol reagent (TaKaRa Otsu, Shiga, Japan) according to the manufacturer's instructions. cDNA was synthesized with the M-MLV reverse transcriptase (Invitrogen, Grand Island, NY, USA) for miR-218 or with the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Otsu, Shiga, Japan) for HPV E6/E7. Real-time PCR was performed in a Real-Time Thermocycler 7500 (Applied Biosystems, Foster City, CA, USA) with a SYBR Green I Real-Time PCR Kit (GenePharma, Shanghai, China) for miR-218 or with a SYBR Premix Ex TaqTM (Tli RNaseH Plus) (TaKaRa Otsu, Shiga, Japan) for HPV E6/E7. For normalization, U6 and β-actin were used as the endogenous controls for miR-218 and HPV E6/E7, respectively. The relative expression levels of miRNAs and mRNAs in each sample were tested in triplicate and were calculated and quantified with the 2^−ΔΔCt method after normalization for expression of the positive control. The oligonucleotide sequences of the miRNA mimics, the miRNA inhibitors, those used for gene knockdown experiments, the gene cloning primers, the 3′UTR cloning primers, and the 3′UTR mutagenesis primers are summarized in Supplementary Table S1.