A pool of two pure subsets of peripheral blood CD4+ T cells (CD19- CD3+ CD4+ CD8- CD16- CD56-), stringently sorted by flow cytometry, from two age and gender matched stage IV (American Joint Committee on Cancer) melanoma patients.
University of Southern California, Norris Comprehensive Cancer Center, Los Angeles, CA
Peripheral blood mononuclear cells (PBMCs) were obtained and cryopreserved in 90% NCS 10% DMSO. Cryopreserved PBMCs were thawed, extensively washed and rested overnight in RPMI 1640 containing 10% FBS, penicillin, streptomycin and L-glutamine. The cells were incubated with fluorescently conjugated monoclonal mouse anti-human antibodies to CD3, CD4, CD8, CD16, CD19 and CD56 (BD Biosciences and Caltag, CA) for 30 mins, washed and resuspended in RPMI 50% FBS and kept on ice. The stained cells were sorted by flow cytometry using the BD FACS Aria into CD8 T cells (CD3+ CD8+ CD4- CD19- CD56- CD16-), CD4 T cells (CD3+ CD4+ CD8- CD19- CD56- CD16-), B cells (CD19+ CD3- CD8- CD4- CD16- CD56-) and CD56dim NK cells (CD19- CD3- CD56dim CD16+). 200,000 cells were sorted into ice-cold RPMI with 50% FBS to preserve cell viability. The sorted cells were pelleted by centrifugation at 1500 rpm 5 mins, homogenized in 1mL TRIzol (Invitrogen, CA) plus 10μg linear acrylamide as a coprecipitant and stored at -80°C. Total RNA was isolated from TRIzol homogenates according to the manufacturer’s protocol and resuspended in RNase-free water. Genomic DNA was removed using the DNA-free kit (Ambion), according to manufacturer’s protocol. RNA quantity and quality were checked using the Nanodrop ND1000A spectrophotometer (Nanodrop Technologies, DE, USA) and the RNA 6000 Pico LabChip assay using the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).
100ng total RNA from sorted cells was used as input for one round of aRNA amplification using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion). Amino-allyl-aRNA samples (2μg of each) were pooled in age and gender matched pairs. The pooled sample was vacuum dried to completion and resuspended in 8μl coupling buffer (Ambion, Inc). aRNA was labeled with 20,000pmol Cy Dye Post Labeling Reactive Dyes (Amersham Biosciences Corp., NJ) at room temperature for 30 mins followed by addition of hydroxylamine to 0.18M to quench unreacted dye, for 15 mins at room temperature protected from light. Labeled aRNA was purified using the RNeasy MinElute Cleanup kit (Qiagen). The labeling resulted in 30-60 dye molecules/1000nt.
Pooled RNA from the total peripheral lymphocyte fraction from 20 healthy donors, used as a common reference.
Stanford Blood Center, Stanford, CA
A total lymphocyte reference RNA was created using the total peripheral lymphocyte fraction from 20 healthy donors (10 male, 10 female) varying evenly in age from 25 to 65 years. PBMCs from these donors were obtained from the Stanford Blood Center. Granulocytes and monocytes were depleted from the PBMCs using RosetteSep depletion cocktails (Stemcell Technologies, Canada). An aliquot of the purified lymphocytes was taken for antibody staining to determine purity. The cells were incubated with fluorescently conjugated monoclonal mouse anti-human antibodies to CD3, CD56, CD16, CD8, CD4, and CD19 for 30 mins, washed, analyzed by flow cytometry (FACS Calibur, BD Biosciences) and were determined to be >99% pure lymphocytes. The remainder of the purified lymphocytes were homogenized in 10mL TRIzol plus 50μg linear acrylamide and stored at -80°C. Total DNA-free RNA was isolated. 100μg total RNA from each of the 20 healthy donor total lymphocyte samples was pooled to create the total lymphocyte reference RNA.
1μg total lymphocyte reference RNA was used as input for one round of aRNA amplification using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion). The aRNA sample was vacuum dried to completion and resuspended in coupling buffer (Ambion, Inc). aRNA was labeled with 20,000pmol Cy Dye Post Labeling Reactive Dyes (Amersham Biosciences Corp., NJ) at room temperature for 30 mins followed by addition of hydroxylamine to 0.18M to quench unreacted dye, for 15 mins at room temperature protected from light. Labeled aRNA was purified using the RNeasy MinElute Cleanup kit (Qiagen). The labeling resulted in 30-60 dye molecules/1000nt.
750ng Cy3-labeled aRNA, 750ng Cy5-labeled total lymphocyte reference aRNA and 50μl Agilent control targets were pooled, and fragmented using the Agilent Fragmentation buffer for 30 mins at 60°C. Agilent hybridization buffer was added, and 490μl of target solution was hybridized onto Agilent Human 1A Oligo Microarrays (v2) using the SureHyb (Agilent) assembly and incubated at 60°C at 4rpm for 17 hours.
The arrays were washed and dried according to the Agilent SSPE wash protocol and scanned using the Agilent Microarray Scanner. The data was extracted from the images using the Agilent Feature Extraction Software, v7.1.
Gene expression profiles of pure sorted peripheral blood lymphocytes from melanoma patients and healthy controls.
The open source R software package (http://www.r-project.org) and tools from the BioConductor project (http://www.bioconductor.org) were used for processing and analysis of the microarray data.
The Agilent control features were removed. Features that were saturated on at least one array were also excluded from analysis. For each array the VSN (variance stabilizing normalization) function was applied to the mean foreground Cy5 and Cy3 intensities, resulting in a ’generalized log ratio’ value for each feature. This was followed by quantile normalization across the arrays.