J82 cells (500,000) were seeded in 60 mm culture plates for adherence and treated with 1:1,100 dilution of frankincense essential oil or 1:11,000 dilution of sandalwood essential oil.
Growth protocol
Human bladder cancer J82 cells were grown in MEM supplemented with 10% FBS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted at 0.5, 1, and 3 hours following treatment using the Rneasy Mini total RNA isolation kit.
Label
Cy3
Label protocol
After purification, RNA concentrations were determined with a scanning spectrophotometer (Nanodrop, Wilmington), and qualitatively assessed for degradation by capillary gel electrophoresis (Agilent 2100 Bionalalyzer, Agilent Technologies). Biotinylated, amplified RNA was produced from 150 ng total RNA per sample using a modification of the Eberwine protocol14 as described in the Illumina® TotalPrep RNA Amplification Kit (Ambion, Inc., Austin). Briefly, RNA was reverse-transcribed with oligo(dT) primers containing a T7 promoter and amplified by in vitro transcription to generate anti-sense RNA containing biotin-UTP ribonucleotides.
Hybridization protocol
After purification, RNA concentrations were determined with a scanning spectrophotometer (Nanodrop, Wilmington), and qualitatively assessed for degradation by capillary gel electrophoresis (Agilent 2100 Bionalalyzer, Agilent Technologies). Biotinylated, amplified RNA was produced from 150 ng total RNA per sample using a modification of the Eberwine protocol14 as described in the Illumina® TotalPrep RNA Amplification Kit (Ambion, Inc., Austin). Briefly, RNA was reverse-transcribed with oligo(dT) primers containing a T7 promoter and amplified by in vitro transcription to generate anti-sense RNA containing biotin-UTP ribonucleotides.
Scan protocol
This RNA was hybridized overnight at 58C to mouse WG-6_v2 Expression BeadChip™ microarrays (Illumina Corp., San Diego), washed under high stringency conditions, labeled with streptavidin-Cy3, and scanned with an Illumina iSCAN scanner.
The images and raw data from each chip were transferred automatically to the microarray database using one of the specified microarray core servers. Non-normalized - Raw signal intensity, background-subtracted using Illumina BeadStudio software. Normalized - log10 values processed using two-step normalization (Dozmorov I, Lefkovits I., Nucleic Acids Res. 2009 Oct;37(19):6323-39).
Differential actions of selective frankincense essential oil versus non-selective sandalwood essential oil induced bladder cancer cell cytotoxicity: A microarray and bioinformatics approach
