5 protocols
nucleic acid extraction protocol
RNA was extracted from the samples using Trizol (Invitrogen) before further purification using the RNeasy MiniElute Cleanup Kit (Qiagen), both according to the manufacturer's protocols.
nucleic acid hybridization to array protocol
This protocol describes Agilent's recommended procedures for hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based one-color gene expression analysis.
nucleic acid labeling protocol
This protocol describes Agilent's recommended procedures for the preparation and labeling of complex biological targets for Agilent 60-mer oligonucleotide microarray-based one-color gene expression analysis.
The samples hybridized on the two distinct arrays were processes separately. Following normalization, significance analysis of microarray (SAM) in the J-Express program (http://jexpress.bioinfo.no) was used for identification of differentially expressed genes. Only genes that changed at least 2.0 fold with FDR under 5.0 were considered as differentially expressed genes in cell lines. For GO analysis, cell adhesion-related GO terms were searched for and a Fischers exact test was performed to obtain nominal p-values.
The prostate cell lines EP156T, EPT1, EPT2 and EPT1B8 were grown in MCDB153 medium (Biological Ind. Ltd., Israel) with 1%, 5%, 5% and 5% fetal calf serum (FCS), respectively, and supplemented with growth factors and antibiotics as described in Ke et al. 2008 and Kogan et al. 2006. The cells were grown in tissue culture plates in a humidified atmosphere containing 5% CO2 at 37C. The medium was changed every third day. DNp63A-FLAG in a pcDNA3 vector was obtained from L. Ellisen, Harvard. The L335 N792 attSfipolylinkIREStdTom plasmid was digested with XhoI and blunt ends were made using T4 DNA polymerase (Fermentas Cat# EP0061) and dephosphorylated with calf intestine alkaline phosphatase (Fermentas Cat# EF0341). pcDNA3DNp63A was excised using BamHI and XhoI, and blunted as above. Ligation was performed using HC DNA Ligase (New England Biolabs, Cat# M0202M). Correct insertion was verified with restriction analysis using SpeI (New England Biolobs, Cat# R0133S) and sequencing. Pseudotyped retroviral particles were prepared in HEK293 Phoenix cells. Retroviral transduction and selection of new cell lines using Fluorescence Activated Cell Sorting (FACS). For p63 knock-down, a pool of three shRNA lentiviral particles (Open Biosystems, clone IDs: V3LHS_397883, V3LHS_397884 and V3LHS_397885) were used, and similarly for ZEB1 knock-down (Open Biosystems, clone IDs: V3LHS_356184, V3LHS_356186 and V3LHS_356187), viral particles with clone ID RHS-4348 were used as negative control. Cells were transduced with a MOI of 1, and subsequently sorted by FACS using the turboGFP reporter in the inserted sequence. The CDH1 gene in a Gateway pDONR vector (Cat# GC-M0954, GeneCopoeia, Rockville, MD, USA) was cloned into pLenti7.3/V5-DEST Gateway Vector Kit (Invitrogen, Carlsbad, CA, USA). The vector was then sequenced to confirm correct insertion. EPT1 and EPT1 DNp63A cells were transduced according to manufacturers protocol and cells expressing CDH1 were selected using FACS based on EmGFP expression.