Sybr luna universal qpcr master mix kit

RNA was extracted using a commercially available kit (Monarch Total RNA Miniprep Kit, New England Biolabs Inc., Ipswich, MA, USA), which enables high-throughput purification of total RNA from up to 96 cultured-cell samples using silica-membrane RNeasy 96 plates. The RNA extracted from cervical cells was used to obtain complementary DNA (cDNA) with reverse transcription, using LunaScript RT SuperMix Kit (New England Biolabs). Quantitative Real-time PCR was performed with Light Cycler 480II (Roche Molecular Biochemicals, Mannheim, Germany) using the SYBR Luna Universal qPCR Master Mix kit (New England Biolabs Inc.). The solution for OCT4, DAZL, Nanog, and G6PD as control gene consisted of 10 μL Master mix, 1 μL of Forward Primer (10 pm/μL), 1 μL of Reverse Primer (10 pm/μL), 3 μL H₂O. The primers used for this trial were provided by Eurofins Genomics. The primers and the hybridization probes used for this trial were provided by TIB-MOLBIOL (Table 1). The qRT-PCR reaction cycling profile was 30 s at 95 °C, 1 cycle, 5 s at 95 °C, and 30 s at 60 °C, 40 cycles. The 2^−ΔΔCT method was used to calculate the relative transcript abundance. All qRT-PCR reactions were repeated twice.

To determine whether the reduction in the endothelial resistance of bEnd.3 cells exposed to MCF7CM was associated with the expression of tight junction proteins, a qPCR was performed to quantify the gene expression of tight junction proteins (Occludin and Claudin-5). The bEnd.3 cells were grown in T75 flasks and chronically exposed to normoxic or/and hypoxic MCF7CM as previously mentioned. Following the exposure, a total RNA was extracted using Tripure isolation reagent (Roche, Ref:11667157001, Ground Floor Liesbeeck House River Park, River Lane, Mowbray, Cape Town, South Africa). The first strand of cDNA was synthesized from total RNA using Transcriptor first-strand cDNA synthesis kit (Roche, No. 04379012001). The resultant cDNA served as a template for real-time PCR amplification using SYBR Luna universal qPCR master mix kit (New England bio labs), and Real-Time PCR System (Applied bio-system real-time-PCR instrument (ThermoFisher Scientific, REF 4484643). To amplify a fragment of claudin-5, occludin, and GAPDH (as house-keeping gene), the primer pairs detailed were used (Table 1). The amplification was conducted at 95 °C for 1 s, followed by 44 cycles of 95 °C for 15 s, 63 °C for 30 s and 95 °C for 15 s. Results were analyzed using the Pfaffl method as described by Pfaffl et al., 2002 [23 (link)].

To determine whether the reduction in the endothelial resistance of bEnd.3 cells exposed to U-87CM is associated with tight-junction proteins, qPCR was performed to quantify the gene expression of tight-junction proteins (Occludin and Claudin-5). The bEnd.3 cells were grown in 75 cm² flasks and exposed daily to normoxic or hypoxic U-87CM, as previously mentioned. Following the treatment of bEnd.3 cells, a total RNA was extracted using TriPure isolation reagent (Roche, Ref:11667157001, Ground Floor Liesbeeck House River Park, River Lane, Mowbray, Cape Town, South Africa). The first strand of cDNA was synthesized from the total RNA using a Transcriptor first-strand cDNA synthesis kit (Roche, No. 04379012001). The resultant cDNA served as a template for real-time PCR amplification using a SYBR Luna Universal qPCR Master Mix kit (New England bio labs) using the real-time PCR system (Applied Biosystems real-time PCR instrument (Thermo Fisher Scientific, REF 4484643)). To amplify a fragment of Claudin-5, Occludin, and GAPDH (as the housekeeping gene), the primer pairs detailed in Table 1 were used. The amplification was conducted at 95 °C for 1 s, followed by 44 cycles of 95 °C for 15 s, 63 °C for 30 sec, and 95 °C for 15 s. Results were analysed using the Pfaffl method, as described by Pfaffl et al., 2002 [30 (link)].