Sybr green real time pcr master mix one step qpcr kit

To validate the results obtained by the microarray, DAVID, and IPA analyses, qRT-PCR was performed twice under at least 10 different experimental conditions. Total RNA (10 ng/reaction) extracted from the CMS and C groups was used in the RNA-direct SYBR Green Real-Time PCR Master Mix: One-step qPCR kit (Toyobo Co. Ltd., Tokyo, Japan). Samples were run in duplicate reactions in 96-well plates. Median threshold cycle values were used to calculate fold changes (FC) between the samples from 2 groups. FC values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. The following temperature profile was used: 30 s at 90°C and 20 min at 61°C for reverse transcription according to the manufacturer’s instructions, followed by 45 cycles of 95°C for 15 s, 65°C for 15 s, and 74°C for 35 s. We used the primers of hepatocyte nuclear factor 4 alpha (Hnf4a) and GAPDH, as shown in Table 1.

RT-qPCR was performed as previously described [19 (link), 23 (link)]. Briefly, total RNA (10 ng/reaction) was used in the RNA-direct SYBR Green Real-Time PCR Master Mix: One-step qPCR kit (Toyobo Co. Ltd.) according to the manufacturer's protocol. Samples were examined in duplicate reactions in 384-well plates. The median threshold cycle values were used to calculate fold changes between the groups. The following cycling conditions were used: 30 s at 90°C and 20 min at 61°C for reverse transcription, followed by 45 cycles of 98°C for 1 s, 67°C for 15 s, and 74°C for 35 s. Fold-change values were normalized to β-actin (Actb) levels using the relative standard curve method. Primer sequences for RT-qPCR are shown in Table 1.