Computational protocol: Deoxyschizandrin, Isolated from Schisandra Berries, Induces Cell Cycle Arrest in Ovarian Cancer Cells and Inhibits the Protumoural Activation of Tumour-Associated Macrophages

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Protocol publication

[…] Total cellular RNA was extracted using Easy Blue® kits (Intron Biotechnology, Seoul, Korea) according to the manufacturer’s instruction. Total RNA (500 ng) was reverse transcribed into first strand cDNA (Amersham Pharmacia Biotech, Oakville, ON, Canada) following the manufacturer’s protocol. The synthesized cDNA was used as a template for polymerase chain reaction (PCR) amplification. Real-time PCR was performed using a SYBR Premix Ex Taq™ Kit (TaKaRa, Kyoto, Japan) and Thermal Cycler Dice RealTime PCR System (TaKaRa). Searching and selection of primer sequences were performed using the Primer-BLAST tool available at the National Center for Biotechnology Information (NCBI) website and the Primer3Plus online tool. The primers used for real-time RT-PCR were as follows: for CD163 sense primer, 5′-AGC AGG GAT GTT GGA GTA GT-3′, and anti-sense primer, 5′-TAA GCT GCT GGC AAA GAA CA-3′, for CD209 sense primer, 5′-CCA GGA TGG TCT CGA TCT CT-3′, for anti-sense primer, 5′-CAG CGA GGA AGA AAC CTA CC-3′, for MMP-9 sense primer, 5′-GGA CGA TGC CTG CAA CGT-3′ and anti-sense primer, 5′-CAA ATA CAG CTG GTT CCC AAT CT-3′, for RANTES sense primer, 5′-GGG TTC GGG AGT ACA TCA AC-3′ and anti-sense primer, 5′-CTG TGT GGT AGA ATC TGG GC-3′, for VEGF sense primer, 5′-ATG GCA GAA GGA GGA GGG CA-3′, and anti-sense primer, 5′-ATC GCA TCA GGG GCA CAC AG-3′, for β-actin sense primer, 5′-CAA ACA TGA TCT GGG TCA TC-3′, for anti-sense primer, 5′-GCT CGT CGT CGA CAA CGG CT-3′. A dissociation curve analysis displayed a single peak. PCRs were performed for 45–50 cycles using the following condition: denaturation at 95 °C for 5 s, annealing at 57 or 59 °C for 10 s, and elongation at 72 °C for 20 s. Mean cycle threshold (Ct) of the gene of interest was calculated from duplicate measurements and normalized with the mean Ct of a control gene, β-actin. […]

Pipeline specifications

Software tools Primer-BLAST, Primer3
Application qPCR
Organisms Homo sapiens
Diseases Neoplasms, Ovarian Neoplasms
Chemicals Oxygen