Stratagene mx300sp

Total RNA was extracted from 6 × 10⁶ cells by using ISOLATE II RNA Mini Kit, according to the manufacturer’s instructions. RNA purity and concentration were determined by comparing the absorbances at 260 and 280 nm. The purified RNA samples were stored in TE buffer at −20 °C until further analysis. cDNA was synthesized from total RNA using the High-Capacity cDNA Reverse Transcription Kit. A sample of 1 ng total RNA was used as a template in a total volume of 10 µL, following the manufacturer’s instructions. Next, gene expression was analyzed by TaqMan probe-based real-time PCR assay. TaqMan probes (Life Technologies, Carlsbad, CA, USA) were used to analyze 4 genes (Bax, Bcl-2, Cas-3, and TP53), and 18S RNA (Life Technologies) was included as the reference gene. qRT-PCR was performed using TaqMan^® Real-Time PCR Master Mix (Life Technologies) and Agilent Technologies Stratagene Mx300SP working on MxPro software. The thermal cycling conditions were as follows: 10 min of polymerase activation at 95 °C, followed by 40 cycles of 30 s denaturation at 95 °C and 60 s annealing/extension at 60 °C. Each sample was run in triplicate. The basal expression level was calculated using the Ct method [39 (link)].

MCF-7 and A549 cancer cells were incubated with AEPS and REPS at an IC₅₀ concentration for 24 h. Gene expression was analyzed using TaqMan Probe-Based Real-Time PCR. TaqMan probes (Life Technologies, Carlsbad, CA, USA) were used to analyze five genes with the following probes: (Hs00608023_m1 (BCL2), Hs00180269_m1 (BAX), Hs00153349_m1 (TP53), Hs00236330_m1 (Fas), and Hs00921974_m1 (TNFSF10), and the reference gene was the 18S rRNA gene (Hs99999901_s1) (Life Technologies, Carlsbad, CA, USA). The RT-qPCR was carried out using the TaqMan™ Gene Expression Master Mix (ThermoFisher Scientific, Waltham, MA, USA) and the Agilent Technologies Stratagene Mx300SP (Agilent Technologies, Santa Clara, CA, USA), working with MxPro software version 4.10. RT-qPCR conditions included 10 min polymerase activation at 95 °C, followed by 40 cycles of 30 s denaturation at 95 °C and 60 s annealing/extension at 60 °C. Samples were run in triplicate. The basal level of expression was calculated according to the Ct method [49 (link)].

Isolation and purification of RNA was performed using total RNA isolation kit (A&A Biotechnology). Subsequently, RNA was transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, California, USA). qRT-PCR was performed using TaqMan^® Real-Time PCR Master Mix (Life Technologies) and Agilent Technologies Stratagene Mx300SP working on MxPro software. TaqMan probes (Life Technologies) were used to analyze 8 genes whose products are essential for DSB repair pathways (BRCA1, LIG3, LIG4, PALB2, PARP1, PRKDC, RAD51, XRCC6), and 18S RNA (Life Technologies) was included as the reference gene. The cycling parameters were 95°C for 10 minutes, 30 cycles of 95°C for 15 seconds and 60°C for 60 seconds.

The cells were incubated for 24 h with 0.75 mg/mL of Rc TR extract. The control cells were grown in the absence of the plant extract. RNA isolation kit (A&A Biotechnology, Gdynia, Poland) was used to isolate RNA according to the manufacturer’s protocol. cDNA was synthesized from the total RNA using the TranScriba Kit (A&A Biotechnology) according to the manufacturer’s protocol. Real-time PCR was performed using TaqMan^® Real-time PCR Master Mix (Life Technologies, Carlsbad, CA, USA) and Agilent Technologies Stratagene Mx300SP with MxPro software. TaqMan probes (Life Technologies) were used to analyze two genes (UHRF1, DNMT1). As the reference gene was used 18S RNA (Life Technologies). The qRT-PCR programme was 95 °C for 10 min, 30 cycles of 95 °C for 15 s and 60 °C for 60 s. Each sample was repeated three times. The comparative Ct method was used to calculate relative fold-changes in gene expression and normalized to the average of 18S RNA.