RNA Extraction and Real-Time qPCR Analysis

Commercial RNA extraction kit (RareRNA, Bio-East Technology, Taipei, Taiwan) was used to isolate cellular RNA, as previously described [11 (link)]. The DNase I kit (Invitrogen, Carlsbad, CA, USA) was used to prepare DNA-free RNA solution. In the experiment, 5 µg of total RNA as starting material, 5 µg of oligo(dT)15 primer (Life Technologies, Carlsbad, CA, USA), and 200 units of reverse transcriptase (Moloney murine leukemia virus; Promega, Madison, WI, USA) were used to synthesize complementary DNA (cDNA) at 42 °C for 45 min. Real-time quantitative polymerase chain reaction (RT-qPCR) amplification was conducted using a standard TaqMan PCR protocol on ABI StepOnePlus system (Applied Biosystems, Foster City, CA, USA). The primers used for RT-qPCR are listed in Table 1. All samples were tested in duplicate wells. The ∆Ct (threshold cycle) was calculated by subtracting of the average glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ct values from the target genes Ct values, which reflects the target gene mRNA level. The change of CBS, CSE, and 3-MST gene expression were calculated as 2^−∆Ct, and the fold change of genes was presented compared to control group.