Housing and care of animals
Female lean (Fa/Fa) and obese (fa/fa) Zucker rats, 5 wk of age, were purchased from the Animal Model CORE Facility of the University of California at Davis. They were housed in the Animal Resource Center of the University of Texas at Austin under standard laboratory conditions. All procedures were approved by the University of Texas at Austin Animal Use Committee.
Tissue samples
Rats were quickly anesthetized by intravenous injection of sodium pentobarbital (50 mg/kg). Red quadriceps muscles from one leg of each animal were removed and freeze clamped as rapidly as possible with tongs that had been cooled in liquid N2. Samples were stored at -70°C for later use.
Extraction of total and messenger RNA
Tissue samples were homogenized in an acid-phenol reagent, with the volume of the tissue not exceeding 10 percent of the volume of the reagent used. Total RNA was then obtained from the homogenate by the procedure recommended by the reagent's supplier (TRI-reagent, Sigma #T9424). This was followed by selection of poly(A) mRNA from the total RNA by hybridizing the RNA with oligo(dT) magnetic microparticles, then isolating the mRNA magnetically (mRNA Isolation Kit, Miltenyi Biotec #751-02). The spectrophotometric 260/280 ratios for mRNA from the obese and lean tissues were 1.95 and 1.99, respectively, which were sufficiently close to 2.0 that an additional round of oligo(dT) selection was not performed.
Preparation of first strand cDNA
Complementary DNA was made using 200 ng of purified, oligo(dT) primed mRNA and SuperScript II RNase H- reverse transcriptase (BRL Life Technologies #18064-014). The cDNA was made radioactive by incorporation of 33P-dCTP (ICN #58201) using the procedure described at http://www.resgen.com/gf200pro.html, except that 33 mM of dATP, dTTP, and dGTP (Pharmacia #27-2035-01) and 1 U/μl of a ribonuclease inhibitor (Ambion #2690) were added. Unincorporated nucleotides were separated from the labeled cDNA using a push column (Stratagene #400701).
Hybridization to array membrane
A high density DNA array membrane was used to measure the level of expression of each of 5,184 genes (Research Genetics, GENEFILTER #GF200, Lot #980611-21). Hybridization of the membrane with radioactive cDNA was performed by the procedures described at http://www.resgen.com/gf200pro.html, except that the last room temperature wash was performed in 2X SSC. The hybridization solution included 0.5 μg/ml poly(dA) (Research Genetics #POLYA.GF) and 0.5 μg/ml cot1 DNA (BRL #15279-011) as blocking agents. Stripping the filter, in order to reprobe it, was done by placing the filter in a boiling 1% SDS solution, as recommended by the manufacturer. The stripped filter was used to expose a phosphorimager plate, which was then scanned. The resulting image had < 0.005 remaining from the image that was obtained before stripping. We obtained nearly identical microarray hybridizations using cDNA prepared from skeletal muscle mRNA from lean (Fa/Fa) versus obese (fa/fa) Zucker rats at an age of 6 weeks (the age at which phenotypic differences between fa/fa and Fa/Fa are thought to begin). Only one gene was possibly expressed differentially (the sarcomeric isoform of the mitochondrial creatine kinase gene, sMtCK, which appeared to have higher expression in the lean muscle). We confirmed the sMtCK result with a Northern blot, but did not do so for any of the other genes, which may be considered a limitation of our experiment.
Quantifying the hybridization results
Radioactivity in hybridized filters was imaged using a phosphorimager (Model 445 SI, Molecular Dynamics). The resulting image files were then processed using custom software, as shown in Fig. 5. Each of the membrane's sixteen 12 × 30-spot arrays was extracted from the original image file, along with a border of pixels corresponding to one spot width. A background value was then estimated for each pixel in each of these images, as follows. If a pixel was situated in the region between or adjacent to DNA spots, and if the spatial derivative of the image at that pixel indicated that it was not part of the overlap region between two intense spots, that pixel was defined to be a background pixel. If a pixel was situated within the region corresponding to a spot of DNA, or if it was situated in the region between two overlapping intense spots, the background value for that pixel was set equal to the value of the nearest background pixel, as defined above. A background-subtracted image of the radioactivity was then obtained by subtracting the background value for each pixel from the original image.
The intensity of spots in an image is a function of how long the phosphorimager plate was exposed, as well as factors such as the efficiency of probe labeling. Therefore, in order to be able to compare background-subtracted images of the same 12 × 30 spot array for successive hybridizations, normalization was performed as follows. The image for one of the hybridizations was selected to be the reference, and corresponding pixels in the other image were normalized by substituting them into a quadratic polynomial normalization function (of the pixel value), the coefficients of which were estimated by singular value decomposition (SVD) [10]. This quadratic model allows for some compensation in the event that the response of the phosphorimager changes between measurements. We found that the fitted second order coefficients were always very close to zero, and the constant offset coefficients were also very close to zero. Thus, normalization of background-subtracted images of the same array for different hybridizations was accomplished essentially by multiplying all pixels in the non-reference image by a constant scale-factor, the value of which had been estimated by SVD. Note that the phosphorimager data have units related to the voltage of its phototube, which are not related in any obvious way to nucleic acid concentrations or amounts. Some investigators rescale the data such that the average signal per microarray spot equals 1, but we did not do so.
The distance between spots on the array is only 0.75 mm, and as a consequence, many of the adjacent intense spots overlap one another. When integrated intensity within a specified region about the center of the spot is used to represent the magnitude of hybridization, it is then useful to model the image as the sum of superimposed two-dimensional spot functions. This was done using two-dimensional gaussian functions to represent the spots, as shown in Fig. 5, with the location, amplitude, and standard deviation coefficients all estimated automatically from the background-subtracted image in two dimensions, using the Levenberg-Marquardt method [10]. The similarity between background-subtracted and fitted spot images (Fig. 5b vs. 5c) indicates that much of the spread of the intense spots appears to be gaussian, which might be attributed to the gaussian shape of the laser beam that scans the phosphorplate. When the spot intensity was close to the noisy background level, this model cannot be used to fit the data, due to near singularity of the matrix equations that had to be solved to perform the fitting. In that case, the method that we used to estimate the intensity of each spot was to sum the nine adjacent pixels in the center of each spot of the background-subtracted image. The correlation between the values so obtained and the corresponding value of the integrated spot intensity from successful gaussian curve fitting was 0.99.
A histogram was constructed from the logarithm of the values of all the spot values. As observed in [11], the histogram for our muscle data was bimodal, consisting of a gaussian-like distribution of low-intensity spots, overlapping an adjacent distribution of moderate and high-intensity spots. The transition between these two distributions occurred within a clearly recognizable range of values, 1550 to 1800 phosphorimager units. We therefore followed the conservative practice recommended in [11] by considering all spot values less than a value of 1800 to define undetectable hybridizations. According to this criterion, 567 of the spots were detectable.
Search for promiscuous hybridization to polyA segments
To investigate the polyA effect as a potential artifact, we performed the following experiment, using essentially the approach described in [4]. Oligo(dT) (10–20 mer mixture, Research Genetics cat. # POLYT.GF) was end labeled with T4 kinase (Life Technologies #10476-018) and (γ-33P)ATP (ICN #58000). The array filter was then hybridized with this probe in Hybrisol I (Oncor #S4040) for 10 hours at 42°C, then washed twice at room temperature for two minutes in 6X SSC and 0.1% SDS. It was then used to expose a phosphorplate for 12 hours, which was scanned using a phosphorimager (Model 445 SI, Molecular Dynamics) to identify the array spots having long stretches of poly(dA). Quantifying of the spot intensities was performed as indicated above.
Staining of the microarray filter with a fluorescent nucleic acid indicator
After all hybridizations of the microarray filter had been performed, we stained the filter with a fluorescent dye that is specific for nucleic acids. We did so in order to determine whether the absence of hybridization signals for some microarray spots was due to the absence of any DNA there, which would indicate a defect in the manufacturing of the microarray at those spots. The filter was stained with 1 μg/ml acridine orange (Sigma A6014) for 10 minutes at room temperature, rinsed in distilled water, then imaged with a fluorimager (Molecular Dynamics). The dye was excited by the 488 nm argon laser line of the fluorimager, and emission was detected with a narrowband filter centered at 530 nm.