Wonder rt cdna synthesis kit

Manufactured by Euroclone

Sourced in Italy

The Wonder RT cDNA synthesis Kit is a laboratory product that facilitates the conversion of RNA into complementary DNA (cDNA) molecules. The kit provides the necessary reagents and enzymes to perform this process, which is a fundamental step in various molecular biology and genomics applications.

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5 protocols using wonder rt cdna synthesis kit

Macrophage Polarization Profiling by RT-qPCR

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Macrophage polarization and anti-inflammatory activity was evaluated by Real-Time Quantitative PCR (RT-qPCR) according to the manufacturer’s protocols. For RT-qPCR, total RNA was extracted from cells through TriFast (EuroClone, Milan, Italy), and cDNA was synthesized using Wonder RT cDNA synthesis Kit (EuroClone). Then, gene expressions of TNF-α, IL-1β, CCL18, CD206, IL-6, IL-12, and IL-10 were evaluated by 7900 HT fast Real-Time PCR System, (Applied Biosystem, Foster City, CA, USA) with SYBR Green PCR Master mix (EuroClone). Gene expression was quantified by the 2^−ΔΔCt method and normalized against 𝛽-actin used as the internal reference gene. Results were expressed as fold changes versus control. Primers used for RT-qPCR are reported in Table S1.

Di Cristo F., Valentino A., De Luca I., Peluso G., Bonadies I., Di Salle A, & Calarco A. (2023). Polylactic Acid/Poly(vinylpyrrolidone) Co-Electrospun Fibrous Membrane as a Tunable Quercetin Delivery Platform for Diabetic Wounds. Pharmaceutics, 15(3), 805.

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Quantification of Antioxidant Gene Expression in Chicken Intestinal Tissue

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Total RNA was extracted from chicken instestinal tissue and conserved in RNA later, by using a Quick-RNA MiniPrep plus kit (Zymo Research, Irvine, CA, USA) following the procedure described by the manufacturer. The concentrations of RNA samples were then quantified by using the NanoDrop 2000 Spectrophotometer (ThermoFisher, USA).
cDNA were synthesized by exploiting the Wonder RT-cDNA Synthesis Kit (Euroclone, Milan, Italy) and amplified by polymerase chain reaction (PCR) in presence of specific primers (Table 1) for SOD, CAT, and β-actin was used as house-keeping gene. The PCR mixture was composed by 1.5 U of a recombinant thermostable DNA polymerase (Wonder Taq Hot Start; Euroclone, Italy), 0.4 μM of each primers and a reaction buffer containing 5 mM dNTPs, 15 mM MgCl₂, stabilizers and enhancers. The amplification was obtained by applying the following thermal program: 95°C for 1 min followed by 30 cycles at 95°C for 15 sec, Tm (melting temperatures reported in Table 1) for 15 sec, 72°C for 30 sec with a final extension at 72°C for 7 min. The PCR products were resolved on 1% agarose gel and the quantitative analysis of visualized spots was performed by using the Gel Doc 2000 software (Bio Rad Laboratories, Hercules, CA, USA).

Ianni A., Bennato F., Di Gianvittorio V., Di Domenico M., Martino C., Colapietro M., Cammà C, & Martino G. (2022). Impact of different shades of light-emitting diode on fecal microbiota and gut health in broiler chickens. Animal Bioscience, 35(12), 1967-1976.

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Quantification of MerTK and ADAM9 Genes

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RNA was extracted from ARPE-19 cells cultured using an RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. The amount and quality of the RNA were determined spectrophotometrically. RNA samples with A260/A280 values of at least 2.0 were used for further analysis. Reverse-transcription of 1 µg of RNA to cDNA was performed using Wonder RT-cDNA Synthesis kit (Euroclone, Milan, Italy). Expression levels of the target genes MerTK (Applied Biosystems, Monza, Italy, assay ID: Hs01031968_m1) and ADAM9 (Applied Biosystems, Monza, Italy, Hs00177638_m1) were measured by qRT-PCR amplification, performed using Luna Universal Probe qPCR Master Mix (New England Biolabs, NEB, Ipswich, MA, USA) in an ABI PRISM 7900 HT Fast Real Time PCR System (Applied Biosystems, Monza, Italy). All reactions were performed in triplicate with the following qRT-PCR run protocol: initial denaturation (95 °C, 1 min), denaturation (95 °C, 15 s), and extension (60 °C, 30 s) repeated 43 times (95 °C, 15 s and 60 °C, 1 min). Gene expression was normalized using β-Actin and GAPDH as control genes (β-Actin assay ID: Hs01060665_g1, GAPDH assay ID: Hs02758991_g1, Applied Biosystems, Monza, Italy). Comparisons of gene expression were done using the 2^−ΔΔCt method [22 (link)].

Puddu A., Ravera S., Panfoli I., Bertola N, & Maggi D. (2022). High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells. International Journal of Molecular Sciences, 23(3), 1144.

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Quantitative Analysis of Gene Expression in SH-SYS5 Cells

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SH-SYS5 cells were seeded and treated as described above. Then, cells were trypsinized and counted. A total of 5 × 10⁶ cells was centrifuged and washed twice with phosphate-buffered saline (PBS). Total RNA was isolated using a RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and integrity was checked on a 2% agarose gel. A quantity of 1 µg of RNA was employed to generate cDNA using the Wonder RT – cDNA- Synthesis Kit (EuroClone, Milano, Italy). cDNA was used to analyze the levels of transcripts by quantitative real-time PCR (qRT-PCR). The Tli RNase H Plus (Diateh labline, Ancona, Italy) and the LightCycler 2.0 instrument (Roche, Basel, Switzerland) were employed. The first gene analyzed was the house-keeping gene actin, and its CT values were used as standards in the ΔΔCT method of results analysis. cDNA samples from SH-SYS5-treated cells were compared to control cells. Samples were analyzed at least in triplicate and the Student’s t-test was used to verify the significance of results (a p-value less than 0.05 was considered significant). Primers were purchased from Sigma-Aldrich, and their sequences and conditions are listed in Table 1.

Rizzardi N., Pezzolesi L., Samorì C., Senese F., Zalambani C., Pitacco W., Calonghi N., Bergamini C., Prata C, & Fato R. (2022). Natural Astaxanthin Is a Green Antioxidant Able to Counteract Lipid Peroxidation and Ferroptotic Cell Death. International Journal of Molecular Sciences, 23(23), 15137.

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Quantifying Epithelial-Mesenchymal Transition Markers

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Total RNA was isolated using the Trizol (ThermoFisher Scientific), according to the manufacturer’s protocol. RNA integrity was confirmed through agarose gel electrophoresis, and RNA concentration and purity were determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA was reversed transcribed using the Wonder RT cDNA Synthesis kit (Euroclone, Italy). The expression of ET-1, CTGF, CYR61, ANKRD1, E-cadherin, N-cadherin, vimentin, ZEB1, and cyclophilin-A mRNA was evaluated by using Luna Universal qPCR Master Mix (New England Biolabs, USA) on QuantStudio 6-Flex (Thermo Fisher Scientific), according to the manufacturer's instructions. The mRNA expression levels were determined by normalizing to cyclophilin-A mRNA expression and expressed as relative mRNA level (2^ΔΔct). Data are presented as means ± SD. Primer sequences are provided in Additional file 2: Table S2.

Sestito R., Tocci P., Roman C., Di Castro V, & Bagnato A. (2022). Functional interaction between endothelin-1 and ZEB1/YAP signaling regulates cellular plasticity and metastasis in high-grade serous ovarian cancer. Journal of Experimental & Clinical Cancer Research : CR, 41, 157.

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