Quantitect sybr green rt pcr

N2a and SH-SY5Y cells were treated with ginsenoside F1 (0, 1, 2.5, 5, and 10 μM) for 24 h at 37 °C and 5% CO₂. Total RNA was extracted from ginsenoside F1-treated or untreated cells using an RNA isolation kit (Takara Shuzo, Kyoto, Japan), analyzed by qRT-PCR using Quantitect SYBR Green RT-PCR (Qiagen, Hilden, Germany) on a CFX96 system (Bio-Rad), and normalized to β-actin. The primers are listed in Supplementary Table S1.

The expression of these genes was quantified by qRT-PCR using QuantiTect SYBR Green RT-PCR (Qiagen, Waltham, MA). Information regarding the primers of MC1R, TYR, KIT, ASIP, TYRP1, OCA2, KITLG, MITF used for the qPCR can be found in Table 1. β-actin was used as housekeeping gene. Quantitative real-time PCR was performed in triplicate on the Stratagene iQ5 system. The 12.5 μL PCR reaction included 6.25 μL SYBR Premix Ex Taq II (TaKaRa, Dalian,China), 0.25 μL (10 p moL / μL) specific forward primer, 0.25 μL (10 p moL / μL) reverse primer, 0.5 μL ROX reference dye, 0.25 μL (10 ng / μL) diluted cDNA and 5.25 μL RNase free water. Cycling parameters were 95°C for 10 min, followed by 37 cycles of 95°C for 15 sec, 57°C for 30 sec and 72°C for 45 sec. Melting curve analyses were performed following amplifications. At the end of the cycles, melting temperatures of the PCR products was determined between 70°C and 90°C. The iQ5 software (Bio-Rad) was used for detection of fluorescent signals and melting temperature calculations. Quantification of selected mRNA transcript abundance was performed using the comparative threshold cycle (CT) method. The difference in abundance of mRNA for the genes was determined by analysis of variance.

Total RNA from fresh-frozen cell line pellets (biological triplicates) and fresh xenograft tumors (3 tumors per cell line) was isolated using QIAzol and the RNAeasy Mini Kit (both from Qiagen, Hilden, Germany). For cDNA synthesis, 1 µg RNA and the QuantiTect^® Reverse Transkription Kit (Qiagen, Hilden, Germany) were used. Quantitative real-time PCR analysis was performed using the QuantiTect SYBR Green RT PCR (Qiagen, Hilden, Germany) on the thermocycler CFX96^TM and the Real-Time System C1000^TM (BioRad, Munich, Germany). Gene-specific primer pairs were: ß-2-microglobuline (b2m)-sense: 5′–GAC TTG TCT TTC AGC AAG GA-3′; b2m-antisense: 5′–ACA AAG TCA CAT GGT TCA CA-3′; icam1-sense: 5′–aag gtg acc gtg aat gtg ct-3′; icam1-antisense: 5′-cgc tgg cgg tta tag agg ta-3′; tacstd2-sense: 5′–TCC CCT TTC GGT CCA ACA AC-3′; tacstd2-antisense: 5′–AAA CGA TCC CGG GTT GTC AT-3′. For data analysis, raw counts were normalized to b2m gene (ΔCt = CT_target – CT_reference). Fold induction to HCT116 cell line was calculated using the 2^−ΔΔCT method with ∆∆Ct = ∆CT_experimental − ∆CT_control. Each sample was analyzed in triplicates (n = 9).

For the relative HMGA2 / ACTB quantification 25 ng total RNA were mixed with SYBR Green, HMGA2 or ACTB specific primers, nuclease free water (Qiagen, Hilden, Germany) and reverse transcriptase according to the “QuantiTect SYBR Green RT-PCR” protocol (Qiagen, Hilden, Germany). The fluorescence of each sample was analyzed in triplicates. As negative controls a non-template and a no-reverse transcriptase control were included. The experiments were performed using the Mastercycler ep realplex (Eppendorf AG, Hamburg, Germany). qRT-PCR conditions were as follows: 30 min at 50°C and 15 s at 95°C, followed by 40 cycles with 15 s at 94°C, 30s at 60°C and 30 s at 72°C. Finally a melting curve analysis was performed to verify specificity and identity of the qRT-PCR products according to the Eppendorf Mastercycler ep realplex instrument instructions. For the comparison of the relative gene expression levels based on the ∆∆CT method the gene expression level of the untreated CT1258 cells was used as calibrator (calibrator expression level was set as 1). Statistical analysis of the qRT-PCR results was done by using the software tool REST 2009, version 2.0.13. A p-value of ≤ 0.05 was considered as statistically significant.

Total RNA from the NHKs or skin tissues was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany), analyzed by qRT-PCR using the Quantitect SYBR Green RT-PCR (Qiagen) on a CFX96 system (Bio-Rad, Hercules, CA, USA), and normalized with GAPDH.