Computational protocol: Selective Estrogen Receptor β Agonist LY500307 as a Novel Therapeutic Agent for Glioblastoma

Similar protocols

Protocol publication

[…] U87 GBM cells were treated with either vehicle or LY500307 (5 μM) for 48 h, and total RNA was isolated using RNAesy mini kit (Qiagen) according to the manufacturer’s instructions. The purity of prepared RNA was determined 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 in duplicates. The combined raw reads were aligned to UCSC hg19, and the genes were annotated by Tophat. Genes were annotated and quantified by HTSeq-__STRONG_START__DESeq pipeline. Differential expression analysis was performed by DEseq and significant genes with at least 1.5-fold change with p < 0.01 were chosen for analysis. The interpretation of biological pathways using RNA-seq data was performed with ingenuity pathway analysis (IPA) software using all significant and differentially expressed genes. To validate the selected genes, reverse transcription (RT) reactions were performed by using SuperScript III First Strand kit (Invitrogen, Carlsbad), according to the manufacturer’s protocol. Real-time PCR was done using SYBR Green (Thermo Scientific) on an Illumina Real-Time PCR system with the following primers. Results were normalized to β-actin transcript levels, and the difference in fold expression was calculated using delta-delta-CT method.Actin-F-5′- GTGGGCATGGGTCAGAAG-3′; Actin-R-5′- TCCATCACGATGCCAGTG-3′ESR2-F-5′- GGC AGA GGA CAG TAA AAG CA -3′; ESR2-R-5′- GGA CCA CAC AGC AGA AAG AT -3′IL24-F-5′- CTTTGTTCTCATCGTGTCACAAC-3′; IL24-R-5′- TCCAACTGTTTGAATGCTCTCC-3′CDKN1A-F-5′- CTGGAGACTCTCAGGGTCGAAA-3′; CDKN1A-R-5′-GATTAGGGCTTCCTCTTGGAGAA-3′;GADD45A-F-5′- GGTGTACGAAGCGGCCAA-3′; GADD45A-R-5′- GCAGGCACAACACCACGTTA-3′PUMA-F-5′- ATGCCTGCCTCACCTTCATC-3′; PUMA-R-5′- TCACACGTCGCTCTCTCTAAACC-3′CTNNB1-F-5′- AAAATGGCAGTGCGTTTAG-3′; CTNNB1-R-5′- TTTGAAGGCAGTCTGTCGTA-3′SFN-F-5′- TGACGACAAGAAGCGCATCAT-3′; SFN-R-5′- GTAGTGGAAGACGGAAAAGTTCA-3′AKT3-F-5′-TGTGGATTTACCTTATCCCCTCA-3′; AKT3-R-5′-GTTTGGCTTTGGTCGTTCTGT-3′PML-F-5′- CGCCCTGGATAACGTCTTTTT-3′; PML-R-5′- CTCGCACTCAAAGCACCAGA-3′CHEK1-F-5′- ATATGAAGCGTGCCGTAGACT-3′; CHEK1-R-5′- TGCCTATGTCTGGCTCTATTCTG-3′CHEK2-F-5′- TGAGAACCTTATGTGGAACCCC-3′; CHEK2-R-5′- ACAGCACGGTTATACCCAGC-3′GADD45B-F-5′- TACGAGTCGGCCAAGTTGATG-3′; GADD45B-R-5′- GGATGAGCGTGAAGTGGATTT-3′WT1-F-5′- CACAGCACAGGGTACGAGAG-3′; WT1-R-5′- CAAGAGTCGGGGCTACTCCA-3′WNT5A-F-5′- ATTCTTGGTGGTCGCTAGGTA-3′; WNT5A-R-5′- CGCCTTCTCCGATGTACTGC-3′SOS2-F-5′- ATGTAGAGGAGCGAGTTCAGAA-3′; SOS2-R-5′- ATGGTAGTCCACTTTGTACCCT-3′FZD5-F-5′- CATGCCCAACCAGTTCAACC-3′; FZD5-R-5′- CGGCGAGCATTGGATCTCC-3′FZD7-F-5′- GTGCCAACGGCCTGATGTA-3′; FZD7-R-5′- AGGTGAGAACGGTAAAGAGCG-3′NTRK3-F-5′- ACGAGAGGGTGACAATGCTG-3′; NTRK3-R-5′- CCAGTGACTATCCAGTCCACA-3′DDR1-F-5′- AAGGGACATTTTGATCCTGCC-3′; DDR1-R-5′- CCTTGGGAAACACCGACCC-3′DDR2-F-5′- GCTATATGCCGCTATCCTCTGG-3′; DDR2-R-5′- ACTCTGACCACTGACTGGAAG-3′MAGI2-F-5′- TCCGGCTCAAGTGTGTCAAG-3′; MAGI2-R-5′- AGGTTGTCACGAATGATTTGCT-3′MRAS-F-5′ TTCCTCATCGTCTACTCCGTC-3′; MRAS-R-5′ AGGATCATCGGGAATGACTCC-3′ […]

Pipeline specifications

Software tools TopHat, HTSeq, DESeq, IPA
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus
Diseases Brain Neoplasms, Glioblastoma, Glioma, Neoplasms
Chemicals Cisplatin, Estrogens, Lomustine