t 95 C for 2 min. Images were scanned and densitometer analysis of the captured image was per formed with BIO 1 D image analysis software. The sig nal intensities of test genes in different samples were normalized to the respective mouse GAPDH signal intensity. DNA selleck chem Ganetespib microarray The focused, immune function targeted DNA microar ray system was constructed using synthesized oligonu cleotide probes as described in our previous study. Briefly, 228 immune function associated genes were selected and grouped into specific cellular immunologi cal functions, such as chemotaxis, antigen processing, maturation and signaling in dendritic cells, apoptosis, and other immune related activities. A number of non immune related, functional genes, and housekeeping genes were also included.

Oligonucleotide probes were designed and synthesized with a length of approximately 50 nucleotides to represent specifically these genes as defined by the U GET program as reported by Iyer et al. and our previous studies. This gene list is now freely available to the pub lic scientific community upon request. A two color CyDye system was used for determining the ratio of gene expression for test control set sample after different treatments. Reverse transcription and first strand cDNA labeling with amino allyl dUTP A total RNA sample was mixed with 3 ul of Oligo dT primer and heated at 70 C for 5 minutes, then allowed to cool for 10 minutes at room tempera ture. The reaction mixture, contain ing 4 ul of 5X first strand buffer, 2 ul of 0. 1 M DTT, 1 ul of a 20X nucleotide mixture, 1 ul AA dUTP and 1 ul of reverse transcription reaction, was incubated at 42 C for 1.

5 h. The reaction was terminated by addition of 2 ul of 2. 5 M NaOH and followed by incubation at 37 C for 15 min. The reaction mixture was neutralized with 10 ul of 2 M HEPES and the synthesized cDNA product was cleaned up with a Microcon purification kit. The cDNA pellet was then speed vacuum dried and resuspended in 15 ul water. Labeling of amino allyl modified cDNA with CyDye Dacomitinib An aliquot of CyDye was resuspended in 15 ul fresh 0. 1 M NaHCO3 pH 9. 0, immediately prior to use in a labeling reaction. One ali quot of resuspended CyDye was then added to one tube of AA dUTP modified cDNA using Cy3 for control samples, and Cy5 for treated samples.

Tubes were mixed by stirring, and incubated at room tempera ture in the dark for 1 hour, after which 15 ul 4 hydroxy lamine was added to each merely coupling reaction, mixed well and incubated at room temperature in the dark, for a further 15 minutes. CyDye labeled cDNA was then puri fied using a DNA purification kit. The allyl modified cDNA pellet was then speed vacuum dried and resuspended in 5 ul water. Hybridization A probe was prepared in fresh hybridization solution consisting of 30% formamide, 5X SSC, 0. 1% SDS, and 0. 1 mg ml of a nucleic acid blocker, human Cot1 DNA. The probe was incubated in solution at 95 C for 5 min utes, then lightly centrifuged for 2 minutes to collect conden