Materials and Methods Control B cell donor and CLL cell patient characteristics Cells from 38 CLL patients were assessed in this study. All patients were enrolled on a tissue banking protocol, #99-224, prior to sample collection. This tissue banking protocol was approved by the Dana-Farber Cancer Institute (DFCI) Institu- tional Review Board, and informed consent was obtained from all CLL patients prior to sample collection. These patients had white blood counts (WBC) between 15.76103 and 265.66103 cells per microliter of blood. 3 of the CLL patients were treated, while the other 35 patients were untreated. Control blood samples were from healthy donors. ZAP-70 status, IgVH mutation, and genomic aberrations were determined as described in Methods S1. The clinical parameters are summarized in Table S1. Control B cell and CLL cell purification Ten mL of heparinized peripheral blood was subjected to Ficoll- PaqueTM PLUS (GE Healthcare, Pittsburgh, PA, USA) density centrifugation. Control B and CLL cells were purified from peripheral blood mononuclear cells (PBMCs) by positive selection with CD19+ selection beads as suggested by the manufacturer (StemCell Technologies Inc, Vancouver, Canada), and then were immediately used for RNA isolation or activation treatment. Control B cell culture and activation B cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (GIBCO, Carlsbad, CA, USA) supplemented with 10% human AB serum (Mediatech, Inc, Herndon, VA, USA), 1% Penicillin-Streptomycin (GIBCO, Aukland, NZ, USA), 1% HEPES (Mediatech, Inc, Herndon, VA, USA), 1% L-glutamine (Mediatech, Inc, Manassas, VA, USA), 50 mg/mL human transferrin (Sigma, St. Louis, MO, USA) and 5 mg/mL insulin (Sigma, St. Louis, MO, USA). The cells were kept in a 37uC incubator with 5% CO2. For CD40 activation, NIH-3T3 CD40L or NIH-3T3 feeder cells were cultured to confluency and then irradiated for 9 K by Cs- irradiator. Irradiated feeder cells were plated in 6 well plates at 26105 cells/mL overnight at 37uC. The cells were washed twice gently with PBS and the B cell medium was added. Purified control B cells were co-cultured with the feeder cells at 37uC for 24 hr or 48 hr. For BCR activation, purified control B cells were adjusted to 16107 cells in 0.1 M phosphate buffered saline (pH 7.2). Goat anti-human immunoglobulin M (IgM) F(ab’)2 (Southern Biotech- nology Associates, Birmingham, AL, USA) at 10 mg/mL was added to untransformed B cells for 20 min. on ice. These cells were transferred to human B cell medium and cultured for 24 hr or 48 hr at 37uC. For other methods of activation, 56106 control B cells were plated in 5 mL of medium containing lipopolysac- charide (LPS) (0.5 ng/mL, Alexis, San Diego. CA, USA), CpG (1 mM, Hycult Biotech, Canton, MA, USA, the sequence of the CpG is 59 tcgtcgttttgtcgttttgtcgtt 39), or polyI:C (5 ng/mL, Invivogen, San Diego, CA, USA) at 37uC for 24 hr or 48 hr. For experiments involving inhibitors for the c-jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase/ERK kinase (MEK), cells was co-cultured with the JNK inhibitor SP600125 (50 mM, A. G. Scientific, Inc., San Diego, CA, USA) or MEK inhibitor PD98059 (50 mM Sigma, St. Louis, MO, USA) for 24 hr before the isolation of RNA for RT-PCR assay. FACS analysis CLL cell purity was tested using anti-CD5-PE and anti-CD19- FITC (BD Pharmingen, San Diego, CA, USA) antibodies. The activation of control B cells was tested using anti-CD69-FITC, anti-CD80-FITC or anti-CD86-FITC (BD Pharmingen, San MicroRNA in Chronic Lymphocytic Leukemia Diego, CA, USA). These antibodies were incubated on ice with the B cells in the presence of PBS with 2% FBS (GIBCO, Carlsbad, CA, USA) for approximately 30 min., the cells were washed with PBS at 4uC for 10 min., centrifuged for 100 g at 4uC, supernatants were aspirated. The cell pellets were re-suspended in 200 mL of PBS and analyzed by FACSCalibur (Becton Dickinson). Total RNA preparation Total RNA from control B or CLL cells was extracted by Trizol method (Invitrogen, Carlsbad, CA, USA). Dried RNA pellets were re-suspended in appropriate volumes of DEPC/ddH2O. RNA was quantitated by O.D.260/280 using spectrophotometry DU640 (Beckman). MiRNA expression profiling We first profiled 435 miRNAs from 22 CLL samples and 1 control B sample by the Luminex method requiring multiple gel extraction steps as described [17]. To expand our sample size, we then chose 43 miRNAs which were predicted to be differentially expressed in CLL from our previous data, 4 miRNAs from published data, and 3 control miRNAs (listed in Table S2) for large scale profiling with the FlexmiR miRNA labeling kit (Luminex, Austin, TX, USA, as described [18]. In this method, 2.5 mg of total RNA was labeled with biotin, and then it was hybridized with locked nucleic acid (LNA)-modified capture probe coupled with beads. After washing away the unbound RNA samples, streptavidin-phycoerythrin (SAPE) reporter molecules were added to the reaction and then the expression of miRNAs was analyzed on Luminex analyzer. Data analysis for miRNA profiling A mock array was generated by averaging the expression of each miRNA in all samples. Next, a linear correction for each sample to the mock array was performed. The data were analyzed using GenePattern software from Broad Institute. MiR-146b-5p was deleted from the analysis due to abnormally high background bead signals. Gene set enrichment analysis We defined a B cell miRNA activation signature as miRNAs that were significantly altered with a t-test p-value of ,0.05 after correction for multiple comparisons in control versus in vitro activated B cells. We performed independent GSEA analysis for upregulated and downregulated miRNA sets to test for enrichment of activation signature miRNAs in control B cells versus CLL profiles as described in [19]. MiRNA-specific RT-PCR assay Total RNA was treated with DNase I (Ambion, Austin, TX, USA), and cDNA synthesis was carried out using miScript Reverse Transcriptase kit (Qiagen, Hilden, Germany). miRNA-specific RT- PCRs were performed using the miScript primer assay according to the manufacturer’s protocol (Qiagen, Hilden, Germany). The miRNA specific primers and the internal control primers were obtained from Qiagen (Hilden, Germany). The relative expression of specific miRNAs was calculated by the DDCt method. A randomly chosen subset of the CLL cell RNAs samples used for Luminex bead-based miRNA expression profiling were used for validation by RT-PCR-based miRNA expression profiling. Statistical methods Associations between miRNA expression and clinical charac- teristics were assessed using the Fisher exact test for binary PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e16956