Sybr green based real time pcr

To detect the expression of miR-541 and other genes in cells or tissues, total RNA was isolated using Trizol (Takara, Dalian, China). RNA integrity and purity were determined as the 260/280 and 260/230 nm absorbance ratios and the 28 S/18S rRNA ratio. cDNA was synthesized using the miRNA 1st strand cDNA Synthesis Kit (AG, UK). The expression of various mRNAs was detected using SYBR Green-based real-time PCR (Takara, Dalian, China) and the ABI StepOne Real-time PCR Detection System (Life Technologies). Gene expression was analyzed using the 2^−ΔΔCT method. U6 was used as an internal control in LX-2 cells and tissues. For detection of the expression of miR-541 in the serum, total RNA from 200 μL of patient serum samples was extracted using a miRNeasy Serum/Plasma Advanced Kit (Qiagen, Germany). Norgen’s microRNA (cel-miR-39) Spike-In Kit (NORGEN, 59,000) was used to provide a quantified synthetic RNA (cel-miR-39) for spike-in during RNA extraction procedures and subsequent normalization in RT-qPCR assays. After reverse transcription of the sample RNA (with spike-in), the level of cel-miR-39 could be determined by subjecting the cDNA generated to qPCR using SYBR Green. The level of expression of serum miR-541 was normalized to the cel-miR-39 transcript level using the 2^−ΔΔCT method. Primer sequences are listed in Supplementary Table 2.