Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes -- Data Supplement - HTML Page - 4923SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Methods
cDNA Microarray Studies.
To confirm selected results obtained from the Affymetrix arrays, a mouse cDNA expression microarray that contained partial cDNA probes for 453 genes was produced using the methodology described by the Stanford/Synteni groups (Table 7) . Partial cDNAs from the 3'-ends of genes were cloned by PCR and sequenced to verify the product . The probe sequences were blasted against the NCBI mouse database to ensure that the partial cDNA sequences did not contain large regions of homology with unrelated genes. Partial cDNAs were PCR amplified, robotically spotted on microscope slides coated with poly-l-lysine, and immobilized by UV-irradiation using a Stratagene UV-crosslinker. To increase the accuracy of the microarray, the partial cDNAs for each gene were randomly spotted eight times, and cDNAs for the control genes cyclophilin, GAPDH, 36B4, and apoB were randomly spotted at least 24 times.

For cDNA microarray hybridizations, the same total RNA used for the Affymetrix arrays was used to make poly(A)⁺ RNA using messagemaker Reagent Assembly (GIBCO/ BRL). For hybridization, amino allyl dUTPs were incorporated into first-strand cDNAs using 2 m g of poly(A)⁺RNA . Monoreactive Cy3 or Cy5 dyes (Amersham Pharmacia) were then coupled to the incorporated amino allyl nucleotides as per the Fair Play Microarray Labeling Kit instructions (Stratagene). Reference and test samples were mixed and hybridized on the microarray as described . Hybridization signals were detected by laser-induced fluorescence for Cy5 and Cy3 using a GenePix 4000A scanner from Axon Instruments (Foster City, CA) and were quantified with gene pix 3.0 software.

Real-Time PCR.
For mRNA changes that were verified using quantitative real-time PCR, the same total RNA described above was used for real-time RT-PCR as described . Specific primers for each gene were designed using primer express software (PE Biosystems, Foster City, CA) . Unpublished primer sets for genes verified by real-time PCR are as follows: aldolase C, 5', 5'-GAGAAGGTCCTGGCTGCTGTATA-3', and 3', 5'-CATGTTGGGCTTGAGCAGAGT-3' (GenBank ID, BC008184); desmosterol reductase, 5', 5'-AGGCAGCTGGAGAAGTTTGTG-3' and 3', 5'-CCTCGCGGTTCATATAGCAATC-3¢ (GenBank ID, NM_053272); and SCD-3, 5', 5'-GTCTTGTATCCGAGGGACCTTCT-3' and 3', 5'-CGCGTTTTCAAAGGTCTATTCTAAC-3' (GenBank ID, NM_024450). The relative amounts of all mRNAs were calculated using the Comparative C_T method (User Bulletin no. 2, PE Applied Biosystems). Cyclophilin mRNA was used as the invariant control.

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