Animal and RNAI protocols
The appropriate Institutional Animal Review Board had approved all studies. To study the effects of RNAI, C57Bl/6J mice retired breeders were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were individually housed with free access to food and water under 12:12 h light-dark cycle (lights on at 07:00 h). Stereotaxic surgery was performed under anesthesia (2.5% 2-2-2-tribromoethanol, 0.015–0.017 ml/g body weight, i.p.). The RNAI constructs were injected bilaterally using a 1 ul Hamilton syringe placed on a stereotaxic frame into the following coordinates from bregma: anterior-posterior -0.12; lateral ± 0.02; vertical -0.6. Double-stranded siRNA particles to reduce expression of AGRP were designed based on Elbashir et al.  (Fig. 1a). The siRNA oligonucleotides were designed from the beginning of exon 1 of both AGRP and GFP genes and ordered gel-purified and annealed from Oligos Etc. (Wilsonville, OR). SiRNA particles were re-suspended in Rnase-free water to a concentration of 1 mM and 0.5 ul was injected over 2 minutes by stereotaxic surgery bilaterally. The pSUPER-RNAI plasmid constructs were re-suspended in lipofectin (Invitrogen Corp. Carlsbad, CA) to a concentration of 1 ug/ul, and 1 ul of the mixture was injected bilaterally over five minutes using the same coordinates as for the siRNA particles. Body weight and food intake was monitored daily.
Metabolic rate was monitored continuously by indirect-calorimetry for three days following surgery. Air from each cage was sampled every five minutes, and oxygen and carbon dioxide concentrations from each sample were measured independently. From the change in oxygen, VO2 or oxygen consumption can be calculated after normalizing to body weight. Heat production can then be calculated using the following formula: Heat (cal/hour) = ((4.33+0.67 × (VCO2/VO2)) × VO2 × (Body weight) × 60). Therefore, the parameter VO2 is normalized to body weight, whereas heat production is not normalized to body weight.
For Northern blot analysis, all mice were sacrificed toward the end of the light period (between 17:00 and 19:00 h) by decapitation after a brief exposure to carbon dioxide. Brains were quickly removed and the hypothalamus was dissected out, frozen on dry ice and stored at -70°C until use. For immunocytochemistry, mice were perfused with cold PBS followed by 4% paraformaldehyde. Brains were removed, post-fixed in paraformaldehyde then preserved in 30% sucrose solution for 48 hours.
Northern blot analysis
Total RNA was extracted from tissue using TRIzol (GIBCO BRL, Gaitherburg, MD). Concentration of the samples were measured by UV spectrophotometer and normalized to 1 ug/ul. Uniform concentration and integrity of the samples were verified by agarose gel electrophoresis prior to Northern blot analysis. Six micrograms of total RNA from hypothalamus was subjected to Northern blot analysis, as described previously, to measure AGRP mRNA [15, 16, 2, 3]. Briefly, RNA was denatured by incubating with glyoxal and dimethyl sulfoxide for 1 hour at 50°C and separated on a 1.5% agarose 10 mM NaPO4 (pH = 7.0) gel for 1 hour at 100 V. The RNA was then transferred to an Immobilon S (Millipore) membrane by capillary elution overnight in 20 × standard saline citrate (SSC) buffer. Membranes were briefly washed in 6 × SSC, baked at 80°C and cross-linked by UV light. The membranes were prehybridized in Ultrahyb buffer (Ambion) at 68°C for 2 hours and hydribized overnight with 3.5 × 106 DPM/ml of probe at 42°C. The blots were probed with single-stranded internally labeled DNA probe as described previously . The membranes were washed twice in 1 × SSC/0.1% SDS solution for 15 minutes at room temperature, followed by 0.1 × SSC/0.1% SDS solution for 15 minutes at room temperature twice and then for 3 hours at 42°C. The membranes were then exposed to phosphoimager screen overnight. The total integrated densities of hybridization signals were determined by phosphoimager (STORM 860, Molecular Dynamics, Sunnyvale, CA). To monitor RNA loading, membranes were re-probed and hybridized with a 32P-labeled probe encoding 18S ribosomal RNA.
Cryopreserved brains were cut to a thickness of 30 um in a cryostat at -20°C and stored in PBS-B buffer (10 mM PO4 in 0.9% NaCl) containing 0.1% sodium azide at 4°C. Immunocytochemistry was performed with a rabbit polyclonal primary antibody to AGRP peptide diluted 1 to 10,000 (Phoenix Pharmaceuticals, Belmont, CA) followed by Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The immunoreactivity was visualized with the Vector VIP kit (Vector Laboratories, Burlingame, CA). Sections were mounted on microscopic slides, air-dried and cover slipped with VectaMount (Vector Laboratories, Burlingame, CA).
Statistical analysis for the Northern blot was performed by student's t-test, using the JMP statistical package implemented on the Macintosh operating system. A p-value of less than 0.05 was considered significant. For the analysis of the metabolic cage data, a two-way ANOVA (time × RNAI construct) was performed using the JMP statistical package. For analysis of food intake and body weight, a two-way ANOVA (time × RNAI construct) followed, where indicated, by student's t-test (p < 0.05 was considered significant) was performed to compare each independent time point.