Ez pcr core reagents

RNA was extracted for RT-PCR and gene arrays with EZ-PCR Core Reagents (Life Technologies, Grand Island, NY), and custom primers were generated (see Table E2 in this article's Online Repository at www.jacionline. org for primers and probes). 16, 24, [34] [35] [36] [37] [38] Expression values were normalized to human acidic ribosomal protein (hARP). Human HGU133Plus2.0 GeneChip probe arrays (Affymetrix, Santa Clara, Calif) were used for gene arrays. 16, 24, [34] [35] [36] [37] [38] Total RNA was extracted with the Qiagen miRNeasy Mini Kit (Qiagen, Valencia, Calif), and DNA was removed with the Qiagen RNAse-free DNAse Set. Total RNA (50 ng) was reverse transcribed and amplified with Ovation Whole Blood Solution from NuGen (San Carlos, Calif). The labeled target was fragmented and hybridized to probe arrays by using the Encore Biotin Module from NuGen. For further details, see the Methods section in this article's Online Repository.

Fresh cryostat sections were stained with Nile red solution (500 mg/mL) in acetone, and images were acquired by using appropriate filters of the Zeiss Axioplan 2 (LSM 510; Carl Zeiss Microimaging, Thornwood, NY), as previously reported. 19 Quantitative RT-PCR and microarray analysis RNA was extracted for RT-PCR with EZ-PCR Core Reagents (Life Technologies, Grand Island, NY), as previously described. 20 Expression levels were normalized to human acidic ribosomal protein. HGU133Plus2.0 microarrays (GSE#107361; Affymetrix, Santa Clara, Calif) were used, as previously described. 11, 20, 21