strain: C57BL/6J sex: Male diet: Chow feeding period: 20 wk sample type: Total Pooled RNA from Chow Fed Mice
Treatment protocol
Samples of WAT, liver, gastrocnemius muscle and heart were obtained, frozen with liquid nitrogen, and stored at -80oC until subsequent use.
Growth protocol
C57BL/6J mice were maintained in a humidity-controlled room with a 12h light/dark cycle. After 3wk of weaning, male littermates were randomly separated into control (n=6) and treatment (n=6) groups, and maintained in microisolator cages. After an acclimatization period of 1wk, animals were given free access to a chow diet (23% protein, 21% fat, and 55% carbohydrate) or a HF diet (15% protein, 59% fat, and 26% carbohydrate) for 20 wk (58R3 – Testdiet; Purina, Richmond, IN, USA). Following the feeding period, animals were fasted for 6h, anesthetized with pentobarbital, and weighed.
Extracted molecule
total RNA
Extraction protocol
Tissue samples were homogenized at short intervals of time and total RNA was isolated using cold TRIzol to prevent RNA degradation (Invitrogen, Ontario, Canada).Specifically, optimized amounts of TRIzol were used for WAT (2mL TRIzol/100mg tissue) and the other tissues (1mL TRIzol/50-100mg tissue), and RNA isolation using Chloroform (EMD Chemicals, Inc., Gibbstown, NJ, USA) was performed twice for WAT. Next, total RNA was purified with wash buffers (RNeasy Mini Kit; Qiagen, Ontario, Canada) and deoxyribonuclease I (DNase I) (RNase-Free DNase Set; Qiagen, Ontario, Canada) according to the manufacturer’s protocol. The quality and quantity of RNA were determined by agarose gel electrophoresis and ultraviolet-visible (UV/VIS) spectroscopy.
Label
Cy5
Label protocol
The FairPlay II Microarray Labeling Kit (Stratagene, La Jolla, CA, USA) was used to prepare cDNA and perform the microarrays. cDNA was synthesized using 10µg of total RNA, purified, and pooled for the control group. Next, pooled control cDNA and cDNA for each treatment subject were fluorescently labeled with an Alexa Fluor (AF) 555 or 647 dye (Alexa Fluor 555 and 647 DNA Labeling Kits; Invitrogen, Ontario, Canada).
strain: C57BL/6J sex: Male diet: High Fat feeding period: 20 wk
Treatment protocol
The FairPlay II Microarray Labeling Kit (Stratagene, La Jolla, CA, USA) was used to prepare cDNA and perform the microarrays. cDNA was synthesized using 10µg of total RNA, purified, and pooled. Next, pooled control cDNA was fluorescently labeled with an Alexa Fluor (AF) 555 or 647 dye (Alexa Fluor 555 and 647 DNA Labeling Kits; Invitrogen, Ontario, Canada). Mouse M7K microarray chip from the Southern Alberta Microarray Facility (University of Calgary, Calgary, AB) were used. The chip itself contains 6,792 70mer oligos designed against the UniGene database for mouse that clusters gene sequences into non-redundant sets. Qiagen/Operon designed and supplied the oligos used on the chip. The labeled cDNA was hybridized at 37oC for 18 h, and scanned via standard procedure. For every treatment sample and its corresponding control, two microarrays were performed in a dye-swap normalization experiment to reduce signal correlation bias and to ensure that both dyes were functional.
Growth protocol
C57BL/6J mice were maintained in a humidity-controlled room with a 12h light/dark cycle. After 3wk of weaning, male littermates were randomly separated into control (n=6) and treatment (n=6) groups, and maintained in microisolator cages. After an acclimatization period of 1wk, animals were given free access to a chow diet (23% protein, 21% fat, and 55% carbohydrate) or a HF diet (15% protein, 59% fat, and 26% carbohydrate) for 20 wk (58R3 – Testdiet; Purina, Richmond, IN, USA). Following the feeding period, animals were fasted for 6h, anesthetized with pentobarbital, and weighed.
Extracted molecule
total RNA
Extraction protocol
Tissue samples were homogenized at short intervals of time and total RNA was isolated using cold TRIzol to prevent RNA degradation (Invitrogen, Ontario, Canada).Specifically, optimized amounts of TRIzol were used for WAT (2mL TRIzol/100mg tissue) and the other tissues (1mL TRIzol/50-100mg tissue), and RNA isolation using Chloroform (EMD Chemicals, Inc., Gibbstown, NJ, USA) was performed twice for WAT. Next, total RNA was purified with wash buffers (RNeasy Mini Kit; Qiagen, Ontario, Canada) and deoxyribonuclease I (DNase I) (RNase-Free DNase Set; Qiagen, Ontario, Canada) according to the manufacturer’s protocol. The quality and quantity of RNA were determined by agarose gel electrophoresis and ultraviolet-visible (UV/VIS) spectroscopy.
Label
Cy3
Label protocol
The FairPlay II Microarray Labeling Kit (Stratagene, La Jolla, CA, USA) was used to prepare cDNA and perform the microarrays. cDNA was synthesized using 10µg of total RNA, purified, and pooled for the control group. Next, pooled control cDNA and cDNA for each treatment subject were fluorescently labeled with an Alexa Fluor (AF) 555 or 647 dye (Alexa Fluor 555 and 647 DNA Labeling Kits; Invitrogen, Ontario, Canada).
Hybridization protocol
A hybridization solution was prepared by mixing DIG Easy Hyb, yeast tRNA and fish sperm DNA in a 18:1:1 ratio; heating to 65 degrees celsius for 2 min and allowing the mixture to cool to room temperature. For each sample, 5 microliters of labeled cDNA was mixed with 60 microliters of the hybridization solution and heated to 65 degrees celsius, then allowed to cool to room temperature. The hybridization mixture was loaded onto array slides, incubated at 37 degrees celsius for 18 h, and scanned.
Scan protocol
Slides were scanned in a Perkin-Elmer ScanArray 5000TM using the Green HeNe 543.5 nm laser for excitation of Cy3 and the Red HeNe 632.8 nm laser for excitation of Cy5. The TIFF scans were imported into QuantArrayTM version 3.0 (Perkin-Elmer) microarray analysis software for spot identification/quantification and background estimation.
Description
Liver tissue extracted after 20 weeks on a high fat diet - Biological replicate 1.
Data processing
Data was input into Gene Traffic DuoTM (Iobion), filtered to flag spots with intensities less than 100 U, and normalized according to the Lowess method. Data was analyzed using Significance Analysis of Microarrays.
