control, Cy3 dye, mixture of total RNA from liver, skeletal muscle, brain, heart, lung, testes, hypothalamus, brown adipose tissue, white adipose tissue, and kidney of C57 mice'
C57 mouse, total liver RNA, high-fat diet, Week9 The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM (Biospec Products, Bartlesville, OK). RNA was isolated according to the manufacturer recommended protocol. Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA. The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA. The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes. The RNA pellet was then dissolved in nuclease-free water (Ambion). Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction. For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%). This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).
