14 of 16 groups were treated with chemopreventive agents starting 3 days before exposure to ECS. NAC were given with the drinking water, wile all other agents were supplemented to the diet.
Growth protocol
Half of 16 groups of adult male Sprague-Dawley rats, were exposed to ECS for 28 days, while the remaining (sham exposed) were kept for the same period of time in filtered air
Extracted molecule
total RNA
Extraction protocol
The lungs were collected, immersed in an RNA stabilizing buffer and frozen at -80°C
Label
Biotin
Label protocol
Five µg of total RNA was reverse-transcribed using a reaction mix containing 1µg of (3'(N)8-(A)12-biotin-(A)12-biotin 5') oligonucleotide primer. The mixture was incubated for 10 min at 70°C and chilled on ice. Four µl of 5X first-strand buffer, 2 volumes of DTT 0,1M, 1µl of 10 mM dNTPs mix and 1µl Superscript II RNaseH reverse transcriptase (200 U/µl) were added. The samples were incubated for 90 min at 37°C. To denature the RNA/DNA hybrids and degrade RNA templates a mixture of 3,5 µl of 0,5M NaOH/ 50mM EDTA were added to 20 µl of first reaction mix. Five µl of 1M Tris-HCl pH7,6 were added to neutralize the reaction mix, and the labeled targets were stored at -80°C until hybridization.
Hybridization protocol
Microarray were hybridized in 6X SSPE/30% formamide at 25°C for 18h, washed in 0,75XTNT buffer at 37 for 40 min, and processed using direct detection of the biotin-containing transcript by streptavidin-Alexa647 conjugate.
Scan protocol
The processed slides were scanned by using an Axon 4000B microarray Scanner, with the laser set at 635 nm and Power 80% and the photomultiplier set at 70 with a scan resolution of 10 µm
Description
none
Data processing
Local background was subtracted from raw data, which were then log transformed, normalized, and analyzed by GeneSpring software version 7.2 (Agilent Technologies, Santa Clara, CA). Expression data were median centered by using the Gene-Spring normalization option, which normalizes both per gene and per array. Quadruplicate data generated for each miRNA were compared among the various experimental groups by volcano-plot analysis, which evaluates both fold variations and statistical significance of differences by ANOVA and Bonferroni multiple testing correction. Global miRNA expression profiles of the various experimental groups were compared by hierarchical cluster analysis and by bidimensional principal component analysis (PCA).
