SUPPLEMENTARY METHODS
Cell Isolation
Human. Venous blood (20 ml) from healthy volunteers was collected in Vacutainer®
collection tubes containing sodium heparin as an anticoagulant (Becton Dickinson,
Mississauga, ON) in accordance with UBC ethical approval and guidelines. Blood was
processed and peripheral blood mononuclear cells (PBMC) were isolated as previously
described11. PBMC at 1 x 106 cells/ml were seeded into 6-well or 24-well tissue culture
dishes (Falcon; Becton Dickinson) at 37°C in 5% CO2. Subsequent to stimulation,
CD14+ human monocytes were isolated from PBMC by direct positive selection by
magnetic separation using Dynabeads CD14 coated with anti-CD14 monoclonal
antibody (Dynal Invitrogen Life Technologies) as per the manufacturer’s instructions.
Panomics cytokine arrays
THP-1 cells were seeded into 6 well plates (2.5 x 106 cells/well) and treated with 60
ng/ml PMA for 18 h. The cells were then rested in DMEM/10% FBS for 24 hrs and then
treated with IDR-1 or LPS for 18 h. The medium was removed and applied to a
Transignal human cytokine antibody array 3.0 (Panomics, MA6150). The arrays were
developed as per manufacturer’s directions.
RNA extraction, amplification and hybridization to DNA microarrays
RNA was isolated from CD14+ monocytes with RNeasy Mini kit, treated with RNaseFree DNase (Qiagen Inc., Canada) and eluted in RNase-free water (Ambion Inc., Austin,
TX, USA) as per the manufacturer’s instructions. RNA concentration, integrity and purity
were assessed by Agilent 2100 Bioanalyzer using RNA 6000 Nano kits (Agilent
Technologies, USA). RNA was (reverse) transcribed with incorporation of amino-allylUTP (aa-UTP) using the MessageAmpII amplification kit, according to the
manufacturer’s instructions, then column purified and eluted in nuclease-free water.
Column purified samples were labelled with mono-functional dyes, Cyanine-3 and
Cyanine-5 (Amersham Biosciences), according to manufacturer’s instructions, and then
purified using the Mega Clear kit (Ambion). Yield and fluorophore incorporation was
measured using Lambda 35 UV/VIS fluorimeter (PerkinElmer Life and Analytical
Sciences, Inc., USA). Microarray slides were printed with the human genome 21K ArrayReady Oligo Set (Qiagen Inc., USA) at The Jack Bell Research Center (Vancouver,
BC, Canada). The slides were processed, hybridized, scanned and quantified as
previously described11.
Analysis of DNA Microarrays
A web-based, semi-automated software was used for the processing of microarray data
(www.pathogenomics.ca/arraypipe) as previously described11. Essentially, evaluation of
slide quality, normalization, selection of differentially expressed genes and statistical
analysis was carried out using the software ArrayPipe (version 1.391). Processing of the
microarray data included flagging of markers and control spots, subgrid-wise background
correction, data shifting, printTip LOESS normalization, and averaging of biological
replicates to yield overall fold-changes for each treatment group as previously
1
described11. Two-sided one-sample Student’s t-test on the log2-ratios within each
treatment group was used for statistical analysis of the data. Data has been submitted to
Array Express under accession number E-FPMI-8.
Quantitative real-time PCR (qPCR)
Differential gene expression identified by microarray analysis was validated using
quantitative real-time PCR (qPCR) using SuperScript™ III Platinum® Two-Step qRTPCR Kit with SYBR® Green (Invitrogen Life Technologies), as per the manufacturer’s
instructions, in the ABI PRISM® 7000 sequence detection system (Applied Biosystems,
Foster city, CA, USA). Briefly, 250 ng of total RNA was reverse transcribed in a 20 l
reaction volume for 50 min at 42C, the reaction was terminated by incubating for 5 min
at 85C and then digested for 30 min at 37C with RNAse H. The PCR reaction was
carried out in a 12.5 l reaction volume containing 2.5 l of 1/10 diluted cDNA template.
A melting curve was performed to ensure that any product detected was specific to the
desired amplicon. Fold changes were calculated after normalization to endogenous
GAPDH and using the comparative Ct method1. The primers used for qRT-PCR are
reported in Supplementary Table 2.
Supplementary Table 2: Sequence of primers (human) used for quantitative real-time
PCR.
Gene
Forward primer (5’-3’)
Reverse Primer (5’-3’)
CXCL1
GCCAGTGCTTGCAGACCCT
GGCTATGACTTCGGTTTGGG
GAPDH
GTCGCTGTTGAAGTCAGAGG
GAAACTGTGGCGTGATGG
IL-6
AATTCGGTACATCCTCGACGG
GGTTGTTTTCTGCCAGTGCC
IL-8
GACCACACTGCGCCAACAC
CTTCTCCACAACCCTCTGCAC
IL-10
GGTTGCCAAGCCTTGTCTGA
AGGGAGTTCACATGCGCCT
IL-19
GAGCCATCCAAGCTAAGGACA
CTTGGTCACGCAGCACACAT
MCP-1
TCATAGCAGCCACCTTCATTC
TAGCGCAGATTCTTGGGTTG
MCP-3
TGT CCT TTC TCA GAG TGG TTC T
TGC TTC CAT AGG GAC ATC ATA
MEK3
ATAGACCCAAGCGCATCACG
GGATAAGGAAATCGGCCTGTG
MEK6
GAAGGCAGATGACCTGGAGC
TCACTGCCATGATCTGCCC
1
Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res. 29: No. 9 e45.
2