iDC generation and RNA preparation
monocytes were isolated from the peripheral blood samples of healthy donors by negative selection using magnetic cell-sorting (Miltenyi, Auburn, CA) and differentiated into immature dendritic cells (iDC) in RPMI/10%FBS containing 1000 U/ml GM-CSF and 1000 U/ml IL-4 (Peprotech, Rocky Hill, NJ) [16
]. Total RNA of monocytes and iDC was isolated using RNAeasy minikit (Qiagen, Valencia, CA).
Affymetrix GeneChip® Microarray studies
The cRNA labeling and hybridizations were performed according to protocols from Affymetrix Inc. (Santa Clara, CA). Briefly, the mRNA in 5 μg of total cellular RNA was converted to double-stranded cDNA using Superscript (Gibco-Invitrogen) with a T7-(dT)24
primer containing T7 RNA polymerase promoter. The cDNA was in vitro
transcribed to biotinylated complementary RNA (cRNA) by incorporating biotin-CTP and biotin-UTP using Enzo BioArray High Yield RNA labeling kit (Enzo Diagnostics, New York, NY). Biotinylated cRNA from each sample was fragmented to approximately 40–100 bases and 10 μg of the fragmented cRNA were hybridized to the Affymetrix human U95 probe array series (A, B, C, D, and E) for 16 h at 45°C with constant rotation at 60 rpm. Following washes, the hybridized chips were sequentially stained with streptavidin-phytoerythrin (Molecular Probes, Eugene, OR), biotinylated goat anti-streptavidin (Vector Laboratories, Burlingame, CA) and another streptavidin-phytoerythrin for signal amplification. After a series of washes, chips were scanned with an argon-ion laser confocal microscope (Hewlett-Packard, Palo Alto, CA) for fluorescence signal detection. All washes and staining procedures were performed on an Affymetrix Fluidics station. The raw expression data derived from Affymetrix Microarray Suite 4.0.1 software gave each transcript an absolute expression level (signal intensity) and a "present" or "absent" call based on the signal/noise ratio. The data were analyzed on two levels. At the detection level, a call of "present" suggests that positive signal is detected for a probe, while a call of "absence" suggests that negative signal is detected for a probe. Gene expression ratio of different samples for each donor was inferred using the PFOLD algorithm [19
] that employs a Bayesian estimation scheme for estimating the fold-change of gene expression and also the significance of the change (P-value). The comparison level analysis of the iDC and monocytes defines a gene as up-regulated if the signal log ratio between the iDC and monocyte samples is larger than 1 (equals a 2-fold increase) and the target sample is present. RNA samples from 3 individuals were analyzed.
The construction and sequencing of subtraction suppression hybridization (SSH) cDNA libraries
SSH libraries were generated using the reagents and protocols provided by Clontech (Clontech, Palo Alto, CA). In one SSH library (H56), the RNA from iDC was used as "tester" and the RNA from monocytes was used as "driver". In another SSH library (H57), the RNA from iDC was used as "driver" and the RNA from monocytes was used as "tester". In both cases, the starting RNA material was a pool of the RNA samples from 3 individuals used in the microarray experiment. RT-PCR analysis of the SSH products showed that the level of the house-keeping gene GAPDH decreased more than 1000 fold in both H56 and H57 cDNA when compared with unsubtracted cDNA (data not shown), suggesting that the subtraction procedure was very effective. 10000 clones from each SSH library were sequenced with M13 primers using the ABI BigDye Terminator v2.0 Cycle Sequencing Kit (Applied Biosystems, Foster City, California) and ABI 3700 DNA Analyzers (Applied Biosystems), according to the manufacturers' protocols and manuals. The SSH cDNA was also used to prepare the cRNA for GeneChip microarrays. In vitro transcription was carried out from the T7 promoter in the PCR primers for SSH. The cRNA generated was used for GeneChip microarray hybridization as described above.
Annotation of the sequence results
Sequences generated through the deep sequencing were clustered into contigs before being submitted to BLAST searches of various online databases to elucidate the identity of clones. These included the National Center for Biotechnology Information (NCBI) nr (nonredundant GenBank, EMBL, DDBJ, and PDB), EST (nonredundant GenBank, EMBL, and DDBJ EST divisions), Incyte LifeSeq® database (Incyte, Palo Alto, CA) and Celera database (Celera, Rockville, MD). The sequences extended in silico were used to search for correspondent qualifiers on the U95 series of GeneChip microarrays (Figure ).
Real-time RT-PCR analysis
The probes and primers used in the TaqMan® (Applied Biosystems, Foster City, CA) analysis are listed in Table . Isolated RNA was treated with Dnase I at 37°C for 1 hour to remove any genomic DNA contamination and first-strand cDNA was then synthesized from the DNased RNA using the ABI Reverse Transcriptase Kit (PE Applied Biosystems, Foster City, CA) in a reaction with RNA at 20 ng/μl. cDNA samples were diluted in TE buffer at 1:20 and plated in triplicate in adjacent wells at 10 μl in a 96-well MicroAmp Optical plate (PE Applied Biosystems, Foster City, CA). Three wells without any template were also included on each plate as negative controls. Real time TaqMan PCR was performed using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). GAPDH was amplified along with the target gene as an endogenous control in each well with VIC-labeled probe to normalize expression between different samples. The probes and primers for target gene and GAPDH were diluted in the TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA) and 15 μl of the reaction mix were added to each well. The probe and primer concentrations in the final 25 μl reaction mix were 250 nM and 900 nM for target gene, and 100 nM and 50 nM for GAPDH. Reactions were performed by an initial incubation at 50°C for 2 min and at 95°C for 10 min, and then cycled at 95°C for 15 sec and 60°C for 1 min for 40 cycles. Output data generated by the instrument on-board software Sequence Detector Version 1.6.3 (PE Applied Biosystems, Foster City, CA) were transferred to a custom designed Microsoft Excel macro for analysis. The differential mRNA expression of each studied gene was calculated with the comparative Ct method using the formula 2ΔΔCt. Here, ΔCt stands for the difference between the target gene and the endogenous control, GAPDH, adjusted by the Ct difference between these 2 genes in negative controls, and ΔΔCt equals to the difference between the ΔCt value of the target gene in iDC and monocyte samples.
Probes and primers used in TaqMan® analysis