Computational protocol: Therapeutic utility of natural estrogen receptor beta agonists on ovarian cancer

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Protocol publication

[…] ES2 cells were treated with either vehicle or Liq (100 μM) for 24 h, and total RNA was isolated using RNAesy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purity of isolated RNA was determined by using Agilent 2100 BioAnalyzer. Illumina TruSeq RNA Sample preparation was performed following manufacturer's protocol, and the samples were run on an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA) in duplicates. RNA-sequencing was performed as described previously []. Differential expression analysis was performed by using DEseq, and significant genes with at least 1.5-fold change with p<0.05 were chosen for analysis. Using all significant and differentially expressed genes from the RNA-seq data, Ingenuity Pathway Analysis software (IPA) was used to interpret the biological pathways. To validate the selected genes, reverse transcription (RT) reactions were performed by using SuperScript III First-Strand kit, according to the manufacturer's protocol and real-time PCR was done using SYBR Green with the following primers: Actin: F-5′- GTGGGCATGGGTCAGAAG-3′; R-5′- TCCATCACGATGCCAGTG-3′ CXCL8: F-5′-ACTGAGAGTGATTGAGAGTGGAC-3′; R-5′-AACC CTCTGCCCCAGTTTTC-3′; MMP14: F-5′-CGAGGT GCCCTATGCCTAC-3′; R-5′-CTCGGCAGAGTCAAA GTGG-3′; MMP11: F-5′-CCGCAACCGACAGAAG AGG-3′; R-5′-ATCGCTCCATACCTTTAGGGC-3′; ICAM1: F-5′-ATGCCCAGACATCTGTGTCC-3′; R-5′-GGGGTCTCTATGCCCAACAA-3′; VCAM1: F-5′-CAG TAAGGCAGGCTGTAAAA GA-3′; R-5′-TGGAGCTG GTAGACCCTCG-3′; MMP1: F-5′-CTCTGGAGTAAT GTCACACCTCT-3′; R-5′-TGTTGGTCCACCTTTCATC TTC-3′; PIK3CG: F-5′-GGCTACCATGAGCAGCTTA CC-3′; R-5′-CTGTGAGGTCGGTGTTCCG-3′; TNF: F-5′-CCTCTCTCTAATCAGCCCTCTG-3′; R-5′-GAGGA CCTGGGAGTAGATGAG-3′; MMP3: F-5′-TTCACTC AGCCAACACTG AA-3′; R-5′-ACAGCATCAAAGG ACAAAGC-3′; IL-1 beta: F-5′-AAGCTGAGGAAG ATGCTG-3′; R-5′-ATCTACACTCTCCAGCTG-3′; PTGS2: F-5′-CTGGCGCTCAGCCATACAG-3′; R-5′-CGCACTTATACTGGTCAAATCCC-3′. Results were normalized to β-actin transcript levels, and the difference in fold expression was calculated using delta-delta-CT method. […]

Pipeline specifications

Software tools DESeq, IPA
Application RNA-seq analysis
Organisms Homo sapiens, Homo sapiens/Mus musculus xenograft
Diseases Neoplasms, Ovarian Neoplasms, Tooth Migration
Chemicals Cisplatin, Estrogens, Paclitaxel