Computational protocol: Potent anti-cancer effects of less polar Curcumin analogues on gastric adenocarcinoma and esophageal squamous cell carcinoma cells

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Protocol publication

[…] Cells were cultured in 6-well plates at a confluence of 2 × 105 cells/well and kept at 37 °C in a moistened air of 5% CO2 overnight. Then, cells were treated for 48 h with respective IC50 values.For RNA extraction from cells, Trizol reagent (Cat. No: 15596-026, Invitrogen, CA, USA) was used according to the manufacturer’s protocol. First-strand cDNA was generated from the cells’ extracted RNA by the RevertAid First Strand cDNA Synthesis Kit, Fermentas (Cat No: #K1621, Maryland, USA) according to manufacturer’s directions.Primers (Bax, cyclin D1, VEGFA, Bcl-2, Caspase 3, c-myc, survivin and the Homo sapiens ribosomal protein L38 (RPL38) as a housekeeping gene) were designed using Primer Express 3.0 (PE Applied Biosystems, Foster City, CA, USA). See Table  for the details of primers used in quantitative real-time PCR. For accuracy and specificity, all primers were blasted in the NCBI website: http://www.ncbi.nlm.nih.gov/tools/primer-blast/. Primers were synthesized by the custom oligonucleotide synthesis service, Metabion (Martinsried, Germany). Quantitative analysis was done using StepOne Real-Time PCR System (Applied Biosystems 7500, Foster City, CA, USA) with the PowerSYBR Green PCR Master Mix (Cat. No: 4309155, Applied Biosystems, Foster City, CA, USA).Individual reaction mix contained an overall volume of 25 μl (master mix 12.5 μl, cDNA 3 μl, primer 3 μl, and H2O 6.5 μl). Thermocycling conditions were as follows: 50 °C for 2 minutes, 95 °C for 10 minutes, then 40 cycles of 95 °C for 30 seconds, 60 °C 30 seconds, and 72 °C for 30 seconds.Relative quantities of target mRNA in test samples were measured and standardized to the housekeeping gene, RPL38 mRNA transcript level. The comparative Ct method was used to assess expression as previously described by Livak and Schmittgen. […]

Pipeline specifications

Software tools Primer Express, Primer-BLAST
Application qPCR