Perfecta sybr green supermix reagent

Total RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel, catalog 740955.250). The cDNA was synthesized by reverse transcription (RT)-PCR from 1 μg of purified RNA with M-MLV reverse transcriptase (Thermo Fisher Scientific, catalog 28025-013) and random primers (Thermo Fisher Scientific, catalog 58875). qPCR reactions were performed in a Viia 7 Real-Time PCR System (Thermo Fisher Scientific) using PerfeCTa SYBR Green SuperMix reagent (Quantabio, catalog 95056-500) and gene-specific primers (Supplemental Table 4). Data were analyzed using QuantStudio version 1.3 (Thermo Fisher Scientific). Relative expression was calculated using the 2^–ΔΔCt method. The RPLP0 gene was used as housekeeping gene.

HCT-116 cells were transfected with DSNs10%, without plasmid (blank) and associating 10 µg of pc(empty), pc(TP53TG1) or pc(mutTP53TG1), as previously described in Section 2.7. After 72 or 96 h of transfection, cells were collected and total RNA was extracted using Total RNA extraction kit (GeneJet RNA purification Kit, Thermo Scientific, Waltham, MA, USA) and cDNA was synthesized subsequently using 1 µg of RNA (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, USA). RNA expression was determined by quantitative RT-PCR (qRT-PCR) was performed using specific primers (Sigma, USA), described in Table 1, using PerfeCTa SYBR Green SuperMix Reagent (Quantabio, Beverly, MA USA) according to manufacturer’s protocol using AriaMx RealTime PCR system (Agilent Technologies, Cheshire, UK). GAPDH was used for the normalization as a house-keeping gene. Relative fold change was calculated as means of 2^−ΔΔt of expression.

RNA was isolated from cells grown in 35 mm dishes using silica columns [33 (link)]. Reverse transcription reactions were performed using qScript cDNA synthesis kit (Quantabio) with 500 ng of RNA per reaction, according to manufacturer’s instructions. The cDNAs were then precipitated with ethanol and used for qPCR with PerfeCTa SYBR Green SuperMix reagent (Quantabio) on a StepOne^™ Real-Time PCR System, according to their recommended procedures.

RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Thermo Fisher). cDNA was produced using the SuperScript III First-Strand Synthesis kit (Thermo Fisher). Real-time PCR was performed using PerfeCTa SYBR Green Super Mix Reagent (Quanta bio) on C1000 Touch Thermal Cycler PCR machine (Bio-Rad). SYP expression was assessed using forward primer 5′-TGCGCTAGAGCATTCTGGG-3′ and reverse primer 5′-CTTAAAGCCCTGGCCCCTTCT-3′.

Total RNA was purified using the NucleoSpin RNA kit (Macherey-Nagel #740955.250), diluted in 50 μL of molecular grade water, and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by reverse transcription from 250 nG-1μG of purified RNA with the M-MLV Reverse Transcriptase (Thermo Fisher Scientific #28025-013) and Random Primers (Thermo Fisher Scientific #58875) in a final reaction volume of 20 μL. The qPCR reactions were conducted in triplicate on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) from 1 μL of cDNA using the PerfeCTa SYBR Green SuperMix reagent (Quantabio #95056-500) and gene-specific primers (Supplementary Table 2). After an initial denaturation at 95°C for 3 min, samples were subjected to 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s. Data were analyzed using the QuantStudio software version 1.3 (Thermo Fisher Scientific). The relative quantification in gene expression was determined using the 2^–ΔΔCt method. The Rplp0 gene (also known as 36b4), coding for the ribosomal protein large P0, was used as an internal control to normalize the data.