Lightcycler r480ii real time pcr instrument

Twelve DEGs and six DEMs were randomly selected from the HC and LC groups, and the qRT-PCR was performed using the extracted total RNA to validate the selected DEGs and DEMs. The LightCycler R 480 II Real-time PCR Instrument (Roche, Basel, Switzerland) was used for the qRT-PCR reactions. The miRNA expression level was determined by a 10-µL PCR mixture which was mixed of 1 µL of cDNA, 0.2 µL of miRNA-specific primer, 0.2 µL of universal primer (Qiagen), 5 µL of 2 × QuantiFast R SYBR R Green PCR Master Mix (Qiagen, Hilden, Germany), and 3.6 µL of nuclease-free water. The mixture was incubated in a 384-well optical plate (Roche) at 95 • C for 5 min, and then performed 95 • C for 10 s with 40 cycles, followed by at 60 • C for 30 s. For mRNA expression, the PCR mixtures, including 2 mL of real-time PCR-ready cDNA, 0.5 mL of specific primer (10 mM), 6.3 mL of SYBR Premix Ex Taq (2 ×), and 2.7 mL of ddH 2 O, for a total volume of 12 mL, were performed 30 s denaturation at 95 • C, followed by 95 • C for 5 s with 40 cycles, and 62 • C for 20 s. The Supplementary Table 1 shown the primers used for these genes and miRNAs. The internal control for normalization was defined by Elongation factor 1a (EF-1a), and the 2 -CT method was performed to calculated the expression levels of relative genes (Livak and Schmittgen, 2001) (link).