Secondary biotin-labeled horse anti-mouse antibody or goat anti-rabbit ...... 0.00. 0.00. 3.48. TM9SF4 transmembrane 9 superfamily protein member 4. 0. 1. 0. 0.
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SUPPLEMENTAL MATERIALS AND METHODS Subjects Institutional review board approval (The Rockefeller University) and written informed consent were obtained before enrolling patients to participate in this study. The study was performed in adherence with the Declaration of Helsinki Principals. Human tissue samples and the assessment of RNA quality All psoriasis lesional skin biopsies were taken from stable large plaque psoriasis patients whose disease ranged from mild to severe. Paired lesional and non-lesional skin samples from four psoriasis patients were obtained by biopsy. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol, with on-column DNase digestion. Extracted RNA was used for microarray analysis comparing gene expression profiles obtained with single and double amplification methods. Samples of paired lesional and non-lesional skin samples from the other three patients underwent preparation for LCM. To assess the quality of sample total RNA prior to LCM, frozen sliced tissue sections from these samples were prepared and total RNA was extracted using the RNeasy Micro Kit (QIAGEN). Extracted RNA was examined using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The remaining RNA was then used to obtain bulk tissue gene expression profiles. Slide preparation and laser capture microdissection Frozen tissue sections were cut at 8.0μm in a -20℃ cryostat and mounted on membrane mounted metal frame slides (Molecular Machines and Industries, Haslett, MI), then stored at -80℃. Slides were stained with Hematoxylin & Eosin (H&E) prior to LCM. LCM was performed following the manufacturer’s protocol for the CellCut system (Molecular Machines and Industries). LCM cells were collected in 100.0μl of RLT buffer (QIAGEN) containing 1% β mercaptoethanol (Fisher Scientific) within 2 hours after starting H&E staining in order to prevent RNA degradation. The samples in RLT buffer were then stored at -80℃ until RNA extraction was performed. RNA extraction and quantification of LCM cells Total RNA of LCM samples was extracted into 10.0μl of RNase free water using the RNeasy Micro Kit. Quantitative RT-PCR (qRT-PCR) for RPLP0/hARP using EZ PCR Core Reagent (Applied Biosystems, Foster City, CA) was performed to calculate the concentration of RNA. Briefly, 1.0μl of total RNA was diluted into 2.0μl of RNase free water. One μl of diluted total RNA was applied into a well with 49.0μl of RT-PCR reaction buffer including 3.0ul of primers and probe mix. Serial dilutions of total RNA (ranging from 2000.0 pg/μl to 1.0 pg/μl) extracted from human skin samples were prepared and used for making the standard curve. The samples were amplified and quantified by 7900HT Fast Real-Time PCR System (Applied Biosystems) using the following thermal cycler conditions: 2 minutes at 50℃; 30 minutes at 60℃; 5 minutes at 95℃; and 45 cycles of 15 seconds at 95℃ followed by 60 seconds at 62℃. Assays were performed in triplicate. 1
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RNA amplification and hybridization Target amplification was performed according to the Affymetrix protocol for one-cycle cDNA synthesis or two-cycle cDNA synthesis. We made a slight modification on two-cycle cDNA synthesis based on a previous report by Kube et al (2007). Briefly, we used SuperScriptIII (Invitrogen, Carlsbad, CA) instead of SuperScriptII. The total RNA/T7-oligo(dT) mix was incubated at 65℃ for five minutes, followed by incubation for one hour at 50℃ for First-Cycle, First Strand cDNA Synthesis. In the second cycle, non-labeled cRNA was incubated with Random Primers for 5 minutes at 65℃ and SuperScriptIII was added to synthesize first-strand cDNA for one hour at 50℃. Labeling of cRNA transcripts with biotin was performed using the GeneChip IVT Labeling Kit (Affymetrix). Fifteen μg of biotin-labeled RNA was fragmented and hybridized to HGU133 A2.0 arrays (Affymetrix), washed, stained, and scanned according to the manufacturer’s protocol. Quantitative RT-PCR on LCM samples First-strand cDNA synthesis was performed using SuperScriptIII and Random Primers (Invitrogen). Two μl of total RNA was mixed with 19.0μl of cDNA synthesis reaction containing 1.0ul of RNAsin (Promega, San Luis Obispo, CA). One μl of the resulting cDNA was used for each quantitative PCR reaction. TaqMan Universal PCR Master Mix (Applied Biosystems) was used for qPCR with the 7900HT Fast Real-Time PCR System. Thermal cycler conditions were as follows: 2 minutes at 50℃, 5 minutes at 95℃, and 45cycles of 15 seconds at 95℃ followed by 60 seconds at 60℃. Samples were analyzed in triplicate. All data were normalized to RPLP0/hARP. Primers and probes used in this study are listed in Table S12. Quantitative RT-PCR on Bulk samples RNA was prepared from nine pairs of lesional and non-lesional skin biopsies. The EZ PCR core reagents (Applied Biosystems) was used for RT-PCR with the 7900HT Fast Real-Time PCR System. Thermal cycler conditions were as follows: 2 minutes at 50℃, 30 minutes at 60℃, 5 minutes at 95℃, and 40cycles of 15 seconds at 95℃ followed by 60 seconds at 60℃. All data were normalized to RPLP0/hARP. Primers and probes used in this study are listed in Table S12. Immunohistochemistry (IHC) Lesional and non-lesional frozen sections were prepared from biopsies from five patients. Three out of 5 were the same patients used LCM analysis. The slides were stained with STAT-1, CCR7, CCL19, CCL21, CCL27, CXCL13, CXCR5, CD20, and CRIP-1. Tissue sections were incubated with primary antibodies at 4℃ over night. Secondary biotin-labeled horse anti-mouse antibody or goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) was amplified with avidin-biotin complex (Vector Laboratories). 3-Amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO) was the chromogen used. Primary antibodies and their dilutions used are listed in Table S13. Immunofluorecent staining (IF) 2
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Frozen sections of psoriasis lesional skin were prepared from biopsies from three patients. Two out of 3 were the same patients used for the LCM analysis. The slides were fixed with acetone and blocked in 10% normal goat serum (Vector Laboratories) for 30 minutes. For CCR7 staining, 10% goat serum containing 10% normal chicken serum was prepared for blocking. Primary antibodies CCR7, CCL19, and CXCR5 were incubated overnight at 4°C and amplified with the appropriate secondary antibody. For co-localization with CCR7, CCL19, or CXCR5, sections were then co-stained overnight with a second primary antibody and amplified with the appropriate secondary antibody (see Table S13) for 30 minutes. Probeset distance to the 3’ end of transcriptome Probe sequences and annotations were obtained from NetAffx (http://www.affymetrix.com/analysis/index.affx). Accessions for RefSeq transcripts were used for distance calculations. In cases where a probe was annotated with multiple RefSeq transcripts, the shortest one was chosen for further analysis. The sequence of the sixth probe from each probeset was blasted against the corresponding RefSeq transcript using the bl2seq program. The alignment coordinates were extracted to where a 100% identity match was found. The probeset distance to the 3' end of the transcript was calculated by subtracting the 5' alignment coordinate from the RefSeq transcript length. Statistical analysis Microarray data was analyzed using GeneSpring GX version10.0 software (Agilent) and R/Bioconductor packages. The Harshlight package (Suárez-Fariñas et al. 2005) was used to scan Affymetrix chips for spatial artifacts. Expression values were obtained using the RMA procedure. Quality Control of the expression values was carried out using the Bioconductor package arrayQualityControl. Expression values were modeled using the mixed-effect framework of Bioconductor’s limma package. Differential expression between lesional and nonlesional skin in each of the groups was assessed using a moderated student’s paired t-test followed by multiple corrections using the Benjamini-Hochberg procedure. FDR2.0) for EPI 0.05 0.1 0.15 0.2 14 647 95 7 573 21 1.06x10-126 5.75x10-6 1.97x10-133 3.63x10-113
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34 680 158 24 622 54 4.30x10-195 1.50x10-18 9.86x10-129 1.96x10-105
41 674 187 32 620 66 3.89x10-229 4.27x10-25 1.67x10-123 5.96x10-99
c. Number of genes by FDR (FCH>2.0) for RD 0.05 0.1 0.15 0.2 97 161 12 2 100 26 5.35x10-11 7.26x10-3 8.77x10-5 7.85x10-11
132 283 26 8 174 49 1.37x10-20 2.00x10-8 1.80x10-13 1.01x10-16
155 341 37 14 211 64 2.72x10-28 4.43x10-14 9.84x10-17 1.32x10-18
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Figure S8. Number of Genes by FDR (FCH>2.0) The numbers of DEGs detected in LCM and Bulk samples are compared across FDR cut-offs with fixed FCH (FCH>2.0), for EPI+RD vs. Bulk (a), EPI vs. Bulk (b), and RD vs. Bulk (c). Corresponding raw data is shown in the table, with results of the Fisher’s exact test and the McNemar test for each FDR. *P2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR2.0 and FDR
