Sybr green pcr master mix

RT-qPCR reactions were prepared in low-profile PCR tubes. For amplification of the cDNA, SYBR Green PCR Master Mix (Themo Fisher Scientific, Oxford, UK) was used according to the manufacturer’s instructions. The protocol for the reactions was set at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 30 s (extension) and melting curve analysis between 65 °C to 95 °C (at 5 s increments). After completing the PCR, the data were exported to an excel file and analyzed in Origin 8.5 statistical software (OriginLab Corp., Northampton, MA, USA).

RNA from indicated cells was extracted by using TRIsure RNA extraction kit (Bioline), and 3 μg of purified RNA was converted into cDNA by reverse transcription with SuperScript III reverse transcriptase (Invitrogene). PCR was performed by using SuperTherm Taq DNA polymerase (GeneCraft) according to manufacturer’s instructions. For Q-PCR analysis, SYBR Green PCR Master Mix (Thermal Fisher) was used according to manufacturer’s instructions. All values were normalized with internal control, GAPDH, and relative gene expression levels were then calculated. Primer sequences are listed in Supplementary Table 1.

Trizol reagent (Life Technology, Milan, Italy) was used to extract total RNA. It was subsequently converted into cDNA through an Applied Biosystem (Foster City, CA, USA) reverse transcription reagent. The quantitative analysis was performed with One-Step Fast Real-Time PCR System Applied Biosystem using the SYBR Green PCR master mix (Life Technology, Milan, Italy). The primer sequences used are shown in Table 2. The mix for PCR analyses included previously synthesized cDNA, SYBR Green PCR master mix (Life Technology, Milan, Italy), primer mix (forward primer/reverse primer) and UltraPureTM Distilled Water DNase/RNase Free (Invitrogen by Life Technologies, Milan, Italy). PCR reactions were subjected to 40 cycles of 95 °C for 20 s, 95 °C for 3 s and 60 °C for 30 s. The relative mRNA expression levels of each gene were determined by the threshold cycle (Ct) value of each PCR product and normalized with GAPDH (Glyceraldeyde-3-phosphate dehydrogenase) by using comparative 2^−∆∆Ct method. The analysis was performed in duplicates.

We developed a melting curve-based allele-specific PCR method to genotype APOE polymorphisms. The method mainly followed the principle of PCR Tm-shift SNP genotyping method described by Wang and colleagues with modification [30 (link)]. In brief, for the genotyping of the codon 112 (rs429358), a 20 μl PCR mixture containing genomic DNA 100 ng, 0.5 μM of each of the APOE-112-F-T, APOE-112-F-C, and APOE-112-R primers, and 1X SYBR Green PCR Master Mix (Life Technologies, CA, USA) was prepared. For the genotyping of the codon 158 (rs7412), a 20 microliter PCR mixture containing genomic DNA 100 ng, 0.5 micromolar of each of the APOE-158-F-C, APOE-158-F-T, and APOE-158-R primers, and 1X SYBR Green PCR Master Mix (Life Technologies, CA, USA) was prepared. PCR was performed with the initial denaturation at 95°C for 5 minutes and then followed by 35 cycles of denaturation at 95°C 1 minute, annealing and extension at 70°C 30 seconds. After PCR, melting curve analysis was performed from 60°C to 95°C with the ramping of 0.3% per minute using the continuous monitor mode. PCR and melting curve analysis were implemented using the Applied Biosystems StepOnePlus Real-Time PCR Systems following the manufacturer’s protocol (Applied Biosystems, Forster City, California, USA). The sequences of the PCR primers and the size of each PCR product are listed in Table 1.

RNA was extracted by Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer instructions. Total RNA was extracted from 2.8 × 10⁶ U937 cells. One μg of total RNA was used to first strand cDNA, which was then synthesized with Applied Biosystem reverse transcription reagent (Foster City, CA, USA).
Quantitative real-time PCR (qRT-PCR) was performed in 7900HT Fast Real-Time PCR System Applied Biosystems using the SYBR Green PCR MasterMix (Life Technologies), using the primer sequences showed in Table S1. qRT-PCR was performed using SYBR Green PCR MasterMix (Life Technology, Milan, IT) 5 μL, Forward Primer 0.2 μL, Reverse Primer 0.2 μL, UltraPureTM Distilled Water DNase/RNase Free (Invitrogen by Life Technologies, Milan, Italy) 4.1 μL, and cDNA 1 μL. The amplified genes were: COX-1 (Ciclooxygenase-1), COX-2, GAPDH (Glyceraldeyde-3-phosphate dehydrogenase), IL-8 (Interleukin-8), and IL-10. The specific PCR products were detected by the fluorescence of SYBR Green, the double-stranded DNA binding dye. PCR reactions were subjected to 40 cycles of 90 °C for 20 s, 95 °C for 3 s and 60 °C for 30 s. The relative mRNA expression level was calculated by the threshold cycle (Ct) value of each PCR product and normalized with that of GAPDH by using comparative 2^-ΔΔCt method. The analysis was performed in triplicate.