strain: B6;129S-Bcmo1-/- Sex: male age: adult treatment: beta-carotene diet tissue: lung, left lobe
Treatment protocol
The experimental setup has been described previously (van Helden et al., CMLS 2010 and van Helden et al., Carcinogenesis 2011). Twelve female and 12 male B6129SF1 (Bcmo1+/+) and 12 female and 12 male B6;129S-Bcmo1tm1dnp (Bcmo1-/-) mice (Hessel et al., JBC 2007) were used for the dietary intervention, which was conducted in accordance with the German animal protection laws by the guidelines of the local veterinary authorities. During the breeding and weaning periods of the mice, mothers were maintained on KLIBA 3430 chow containing 14,000 IU vitamin A/kg diet (Provima Kliba AG, Kaiseraugst, Switzerland). Five-week-old female and male Bcmo1+/+ and Bcmo1-/- mice were caged in groups containing two to four siblings per group and were maintained under environmentally controlled conditions (temperature 24 C, 12 h/12 h light/dark cycle). Mice had ad libitum access to feed and water. Basic feed consisted of the pelletized diet D12450B (Research Diets Inc, USA) with a fat content of 10%. The diet was modified to contain 1,500 IU vitamin A/kg of diet, which is a vitamin A-sufficient diet, and the control diet (control) was supplemented with water-soluble vehicle beadlets (DSM Nutritional Products Ltd., Basel, Switzerland) containing L-alpha-tocopherol and ascorbyl palmitate as stabilizers, as well as carriers such as gelatin, corn oil, sucrose and starch. The BC diet (BC) was supplemented with identical water-soluble beadlets containing BC (DSM Nutritional Products Ltd., Basel, Switzerland) to generate 150 mg BC/kg diet. Beadlets were added by the manufacturer before low temperature pelletting. Feed pellets were color marked and stored at 4 C in the dark. After 14 weeks of dietary intervention, male and female mice were anesthesised using isoflurane and ketamin and killed. After sacrifice, lungs were immediately dissected and snap frozen in liquid nitrogen and stored at -80C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from left lung lobes using TRIzol Reagent (Sigma-Aldrich) according to the manufacturer’s instruction. After extraction, RNA was purified by using RNeasy columns (Qiagen). RNA concentration and purity were measured using the NanoDrop spectrophotometer (IsoGen Life Science). The integrity of RNA was checked with Experion automated electrophoresis system (BioRad).
Label
Cy5
Label protocol
1,000 ng of purified individual total RNA was used for cDNA synthesis, split into two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent linear amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010). Cy-3 labelled samples were pooled on an equimolar basis and served as reference pool; this comprised all samples.
strain: pooled B6129SF1 and B6;129S-Bcmo1-/- Sex: pooled male and female age: adult tissue: lung, left lobe sample type: reference
Treatment protocol
The experimental setup has been described previously (van Helden et al., CMLS 2010 and van Helden et al., Carcinogenesis 2011). Twelve female and 12 male B6129SF1 (Bcmo1+/+) and 12 female and 12 male B6;129S-Bcmo1tm1dnp (Bcmo1-/-) mice (Hessel et al., JBC 2007) were used for the dietary intervention, which was conducted in accordance with the German animal protection laws by the guidelines of the local veterinary authorities. During the breeding and weaning periods of the mice, mothers were maintained on KLIBA 3430 chow containing 14,000 IU vitamin A/kg diet (Provima Kliba AG, Kaiseraugst, Switzerland). Five-week-old female and male Bcmo1+/+ and Bcmo1-/- mice were caged in groups containing two to four siblings per group and were maintained under environmentally controlled conditions (temperature 24 C, 12 h/12 h light/dark cycle). Mice had ad libitum access to feed and water. Basic feed consisted of the pelletized diet D12450B (Research Diets Inc, USA) with a fat content of 10%. The diet was modified to contain 1,500 IU vitamin A/kg of diet, which is a vitamin A-sufficient diet, and the control diet (control) was supplemented with water-soluble vehicle beadlets (DSM Nutritional Products Ltd., Basel, Switzerland) containing L-alpha-tocopherol and ascorbyl palmitate as stabilizers, as well as carriers such as gelatin, corn oil, sucrose and starch. The BC diet (BC) was supplemented with identical water-soluble beadlets containing BC (DSM Nutritional Products Ltd., Basel, Switzerland) to generate 150 mg BC/kg diet. Beadlets were added by the manufacturer before low temperature pelletting. Feed pellets were color marked and stored at 4 C in the dark. After 14 weeks of dietary intervention, male and female mice were anesthesised using isoflurane and ketamin and killed. After sacrifice, lungs were immediately dissected and snap frozen in liquid nitrogen and stored at -80C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from left lung lobes using TRIzol Reagent (Sigma-Aldrich) according to the manufacturer’s instruction. After extraction, RNA was purified by using RNeasy columns (Qiagen). RNA concentration and purity were measured using the NanoDrop spectrophotometer (IsoGen Life Science). The integrity of RNA was checked with Experion automated electrophoresis system (BioRad).
Label
Cy3
Label protocol
1,000 ng of purified individual total RNA was used for cDNA synthesis, split into two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent linear amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010). Cy-3 labelled samples were pooled on an equimolar basis and served as reference pool; this comprised all samples.
Hybridization protocol
Individual Cy5-labelled samples were hybridized against the Cy3-labelled reference pool according to the manufacturers' procedure using Agilent In Situ Hybridization Kit Plus and GEx Hybridization Buffer HI-RPM. Samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers and hybridized at 65C for 17 hours at 4rpm rotation. After hybridization, slides were washed sequentially following Agilents' recommendations.
Scan protocol
Scanned with an Agilent Technologies Scanner G2505B with 10 and 100% laser-power intensities.
Description
Cy3 samples were pooled on an equimolar basis and served as reference pool, and individual Cy5-labelled samples were hybridized against the reference pool.
Data processing
Images were quantified using Agilent Feature Extraction Software (v 9.5.3.1) to obtain raw median signal and background values for both Cy5 and Cy3. Spots with a mean signal higher than twice the background value over all arrays in both channels were considered to be expressed. Data was normalized according to Pellis et al., (Physiol Genomics 2003; 16:99-106) based on the Cy3-reference pool, and log2 transformation.
Nutritional effects by beta-carotene versus control in lung, inguinal white adipose tissue, and liver in males and females of control wildtype mice versus BCMO/BCO1 knockout mice
Data table header descriptions
ID_REF
VALUE
Normalized log2 value of sample over reference pool
