Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Methods Cells in culture The MDA-MB-231 (MB-231) and MCF-7 cell lines were obtained from American Type Culture Collection (Man- assas, VA). The cell lines were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. MB-231 cells were grown in RPMI (GIBCO®, Invitrogen™, Carls- bad, CA) containing 10% fetal calf serum (Hyclone, Thermo Fisher Scientific, Waltham, MA), 0.5 mM L- glutamine (GIBCO®), 25 mg/ml glucose (Sigma-Aldrich), HEPES (GIBCO®) and sodium pyruvate (GIBCO®). MCF-7 cells were grown in DMEM (GIBCO®) supple- mented with 10% fetal calf serum. HuR Immunoprecipitations (RIP-Chip) HuR RIP-Chip was performed as previously described [8,47,48]. Briefly, lysates were prepared from exponen- tially growing MB-231 and MCF-7 cells. Equal amounts of protein lysates were used (100-300 μg). HuR mono- clonal antibody 3A2 (made in our laboratory from the 3A2 hybridoma, generously provided by Dr. Joan Steitz, Yale University, New Haven, CT), or isotype control IgG1 (BD Biosciences, San Jose, CA), were pre-coated onto Protein A Sepharose beads (PAS) and extensively washed. Lysates from each cell initially were pre- absorbed with 30 μg of IgG1 and then removed by addi- tion of PAS beads. Individual pull-downs were per- formed at 4°C for only 1-2 hr to minimize potential re- assortment of mRNAs. RNA amplification The entire amount of recovered RNA per immunopreci- pitation was amplified using the WT-Ovation™ Pico RNA Amplification System protocol (NuGen, San Car- los, CA). Forty ng of total RNA was used as starting material to generate at least 6 μg of cDNA. Amplified cDNA was purified using Zymo Research Clean and Concentrator™-25 (Zymo Research, Orange, CA). Three μg of amplified and purified cDNA was incubated at 50° C for 30 minutes with 5 μl of UNG buffer and 5 μl UNG enzyme and 60 minutes with 5 μl labeling buffer and 5 μl ARP (biotin) solution as described in NuGen’s labeling protocol for the Illumina BeadArray platform. All samples (total RNA, amplified cDNA, and biotin labeled amplified cDNA) were quantitated using a Nanodrop™ (Thermo Fisher Scientific, Waltham, MA) spectrophotometer. RNA quality and integrity were assessed on selected samples with the Experion™ auto- mated electrophoresis system (Bio-Rad, Hercules, CA). Microarray Biotin-labeled, amplified cDNA (1.5 μg) was hybridized to a Sentrix® Human-6 v.2 Whole Genome Expression Page 3 of 15 BeadChips (Sentrix Human WG-6; Illumina, San Diego, CA). Each chip tested 6 samples and contained 47,293 gene targets, representing 18,025 distinct RefSeq genes that are not pseudogenes. A total of 3 chips were used for this experiment. The chips were hybridized at 48°C for 20 hr in the hybridization buffer provided by the manufacturer. After hybridization, the chips were washed and stained with streptavidin-C3. The chips were scanned on the BeadArray Reader, as described by Illumina at http://www.illumina.com. The Illumina BeadStudio software was used to assess fluorescent hybridization signals. Quantitative RT-PCR Selected genes were validated by quantitative RT-PCR. Briefly, cDNA was generated from the same samples as previously described for the microarray experiments using 10 ng total RNA and the SuperScript™ III Plati- num® Two-Step qRT-PCR Kit with SYBR® Green (Invi- trogen Carlsbad, CA). RT-PCR was performed on the StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA). Each sample was run in triplicate for these genes and the cDNA was divided equally per reac- tion in a 20 μl volume. The PCR conditions were: 50°C for 2 minutes and 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds alternating with 60°C for 30 seconds. Melting curve analysis was performed on every reaction to confirm a single amplicon. For each cell line, differences in gene expression were determined using the equation 2-ΔΔCt, where the Ct value for either the HuR or IgG IP was subtracted from the Ct value of the GAPDH control to yield the ΔCt value. For each cell line, the ΔCt value for the HuR and IgG IP were com- puted in triplicate and averaged to give one ΔΔCt value per sample. Primers used: Human RT GAPDH Forward 5’ AGCCTCAAGAT- CATCAGCAATGCC 3’ Reverse 5’ TGTGGTCATGAGTCCTTCCACGAT 3’ Human RT HuR Forward 5’ ATGAAGACCA- CATGGCCGAAGACT 3’ Reverse 5’ AGTTCACAAAGCCATAGCCCAAGC 3’ Human RT CD9 Forward 5’ TCAGACCAAGAG- CATCTTCGAGCA 3’ Reverse 5’ ACCAAGAGGAAGCCGAAGAACAGT 3’ Human RT CALM2 Forward 5’ CTGACCAACT- GACTGAAGAGCAGA 3’ Reverse 5’ TTCTGTGGGATTCTGCCCAAGAG 3’ Cloning strategy of HA HuR Hemagglutinin (HA) tagged human HuR was cloned into the NheI and XhoI sites of the pZeoSV2 (-) vector (Invitrogen). The plasmids were sequenced in both directions to confirm identity. Cells were transfected