PLoS One. 2012; 7(6): e38716.
mRNA Microarray ExperimentThe samples used for mRNA microarray were the same as the miRNA microarray. Microarray was performed by a service provider (CapitalBio, China). The microarray (Probe length 60-mer, 135K Format) containing 44987 probe sets was provided by Roche-NimbleGen. For each sample, 1 µg of total RNA was treated with the CapitalBio cRNA Amplification and Labeling Kit (CapitalBio, China) according to the manufacturer’s instructions. After reverse transcribed with a T7 oligo(dT) primer, second-strand synthesis and purification, the generated cDNAs were in vitro transcribed to synthesize multiple copies of cRNAs. Then 5 µg of purified cRNAs were reverse transcribed with random primer. Labeled cDNA molecules were generated by subsequent Klenow Fragment Polymerase labeling with Cy3-dCTP (GE Healthcare, USA). Following purification and drying, the labeled cDNAs were then dissolved in 80 µl hybridization buffer (3×SSC, 0.2%SDS, 5×Denhart’s, 25% formamide). Hybridizations were performed overnight at 42°C using hybridization system 12 (Roche NimbleGen, USA). The arrays were then washed and dried. The fluorescence intensity was collected using NimbleGen MS 200 Microarray Scanner. Data were extracted from scanned images using NimbleScan v2.6 software. Quantile normalization RMA (Robust Multi-Array) analysis was performed to generate gene expression values. The differences between the two groups were analyzed using SAM (Significance Analysis of Microarrays) method with SAMR software version 3.02 [42]. The FDR (False Discovery Rate) were calculated. Differentially expressed genes (DEGs) were selected with FDR <5% and FDR <10%. All data were MIAME compliant and have been deposited in GEO (accession number GSE33524).
Am J Pathol. 2012 Mar; 180(3): 1189–1201.
IHC Staining Tissue microarray slides containing 285 formalin-fixed, paraffin-embedded breast cancer tissues were used to detect ERRF protein expression by IHC staining. In addition, 54 formalin-fixed, paraffin-embedded noncancerous breast tissues were used to monitor ERRF expression in noncancerous breast cancer tissues. Briefly, after deparaffinization in xylene and rehydration in a series of alcohols (100 − 75%), slides were incubated in the dual endogenous enzyme block (Dako, Carpinteria, CA) for 15 minutes to inactivate endogenous peroxide activity and were treated in citrate buffer (pH 6.0) for 3 minutes in a pressure cooker for antigen retrieval. After cooling for 45 minutes at room temperature, slides were incubated with rabbit anti-ERRF antibody (HPA026676; Sigma-Aldrich, St. Louis, MO) at 1:600 dilution at 4°C overnight and with the secondary horseradish peroxidase–labeled polymer anti-rabbit Igs (Dako) for 30 minutes at room temperature. With diaminobenzidine tetrahydrochloride (Dako) as a chromogen, slides were counterstained with hematoxylin. Preimmune serum was used as the negative control for ERRF antibody. The specificity of ERRF antibody was evaluated by IHC staining of T-47D (ERRF-positive) and MCF-7 (ERRF-negative) breast cancer cell lines prepared in paraffin blocks (see Supplemental Figure S1 at http://ajp.amjpathol.org). Slides with IHC staining were examined independently by two investigators (X.F. and S.F.), and any discrepancy in the reading for a case was discussed and resolved. The intensity of nuclear staining (0 = negative, 1 = low, 2 = medium, and 3 = high) and the percentage of positively stained cells (0 − 100%) were recorded for each specimen, and ERRF expression was expressed as the multiplied score, which was calculated as intensity score × percentage for positive cells × 100. The average multiplied score for the 54 noncancerous breast tissues was ∼15. In addition, the median of ERRF expression levels in primary tumors was 15. Therefore, the cutoff point of 15 was used to classify primary tumors into two groups. Those with a multiplied score <15 were defined as lower ERRF expression, and those with a multiplied score ≥15 were defined as higher ERRF expression. We also used the receiver operating characteristic curve to determine the optimal cutoff points for ERRF expression. In this analysis, the optimal cutoff point is defined when the Youden Index, the potential effectiveness of a biomarker, achieved the maximum.21 With the Youden cutoff point, which varied among different variables, tumors were regrouped as ERRF higher or ERRF lower, and statistical analysis was performed again for each variable.
Oncotarget. 2015 Mar 12
Microarray analysis Total RNA was extracted from Panc1 cells by using a mirVanaTM miRNA Isolation Labeling Kit (Ambion Inc.). The total RNA was quantified by using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technology). The total RNA samples with adequate RNA quality index (>7) were used for microarray analysis; 700 ng of total RNA was used for labeling and hybridization on HumanHT-12 v4 expression beadchip (Illumina, Inc.) according to the manufacturer’s protocols. After the beadchips were scanned with a BeadArray Reader (Illumina), the microarray data were normalized using the quantile normalization method in the Linear Models for Microarray Data (LIMMA) package in the R language (http://www.r-project.org). BRB-ArrayTools were primarily used for statistical analysis of gene expression data, and the Student’s t test was applied to identify the genes significantly different between 2 groups when compared. Differentially expressed genes were identified using > 1.5 or 2 fold change cut off. Gene ontology enrichment analysis was carried out using David Functional Annotation Resources 6.7 (http://david.abcc.ncifcrf.gov/). Data for gene expression study of pancreatic ductal adenocarcinoma were downloaded from Gene Expression Omnibus (GEO, NCBI) (http://www.ncbi.nlm.nih.gov/geoprofiles/).
PLoS One. 2013; 8(2): e57709.
Gene Expression Microarray AnalysesIn parallel with methylation profiling, we performed microarray gene expression analyses following the manufacturer’s instructions and a previously established protocol [39]. Total RNA was isolated using the RNeasy mini-kit (QIAGEN, Valencia, CA) and amplified using Illumina’s TotalPrep RNA Amplification Kit. RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Labeled cRNA was hybridized overnight to Human HT12 V3 chips, washed, and scanned on an Illumina BeadStation-500. Illumina’s BeadStudio version 3.1 was used to generate signal intensity values from the scans, subtract background, and scale each microarray to the median average intensity for all samples (per-chip normalization). Normalization was done using quantiles with the Lumi R-package. Fold-changes were calculated with respect to their respective controls as described elsewhere [35]. Ingenuity pathway analysis (IPA, Ingenuity Systems, Inc. CA, USA) was performed to evaluate und categorize differentially expressed genes into various functional pathways.
PLoS One. 2013; 8(2): e57709.
Quantitative Methylation AnalysesWe validated methylation microarray data by independent single gene/promoter quantitative methylation assays. Depending on the methylation assay design features, we used either bisulfite pyrosequencing (PSQ HS 96A pyrosequencing system, Qiagen, Valencia, CA) or real-time based qMSP for quantitative methylation analyses as described previously [37], [38]. Primer sequences used for both methods are provided in Supplementary Table S1.
PLoS One. 2013; 8(2): e57709.
DNA Extraction, Bisulfite Modification and Methylation Profiling (Infinium Methylation Assay)DNA extraction was performed with the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Genomic DNA was bisulfite modified (EZ DNA methylation Gold Kit, Zymo Research, please add city/state) as described previously [35]. To analyze the effect of curcumin on DNA methylation, we performed DNA methylation profiling using the Infinium methylation assay with HumanMethylation27 BeadChip microarrays (Illumina, San Diego, CA), which are capable of simultaneously analyzing the methylation status of 27,578 individual CpG sites covering over 14,000 genes [36]. Whole genome amplification, labeling, hybridization and scanning were performed according to the manufacturer’s instruction at the genomics core facility at the Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX. Methylation status was measured as the ratio of signal from a methylated probe relative to both methylated and unmethylated probe signals. Methylation ratios were extracted using the Methylation Modules in the Illumina Bead Studio following average normalization, and the quantitative β-value ranged from 0 (0% methylation) to 1 (100% methylation). The p-value cut-off for detectable probe signals was set at 0.05. For the methylation analyses, a Δβ-value of ≥0.1 (10% methylation) was defined as significant [37].
PLoS One. 2013; 8(9): e73785
Microarray analysisTotal RNAs from the colon of three control and three HDAC1/2 IEC-specific knockout mice were isolated with the Rneasy kit (Qiagen, Mississauga, ON, Canada). cDNA preparation and microarray assay were performed at the Microarray platform of the McGill University and Genome, Quebec Innovation Centre. An Affimetrix GeneChip mouse genome 430 2.0 array, displaying over 34,000 murine gene sequences, was used for hybridization. Data analysis, normalization average difference and expression measurements were subsequently completed with Flexarray software version 1.6.1. Background correction and normalization were assessed with a multi-array average (RMA) algorithm. Significant statistical differences were calculated with Welch’s t test, with the cut-off for statistical significance set to a p value below 0.05. Classification of genes according to their Gene Ontology biological processes was performed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.7 (http://david.abcc.ncifcrf.gov/) [26] and the ToppGene suite for functional gene enrichment analysis and candidate gene priorization (http://toppgene.cchmc.org/) [27]. Both analysis tools gave similar results regarding biological processes, and the highest gene count and lowest p value were selected. Microarray data have been deposited in the Gene Expression Omnibus database (GSE47745).
PLoS One. 2011; 6(6): e20656.
Crithidia mellificae strain SF - Microscopy, Culturing and DNA PurificationHoney bees were collected from a San Francisco, CA (U.S.A.) colony previously identified to be Crithidia positive by microarray and PCR testing. Honey bees were immobilized by chilling at 4°C for 20 minutes, briefly washed in 70% ethanol, and decapitated prior to dissection. The SF strain was isolated from honey bee intestines dissected in a sterile environment, minced and placed in a T25 flask and cultured in BHT medium composed of Brain Heart Infusion (BHI) 28.8 g/L (DIFCO), tryptose 4.5 g/L (DIFCO), glucose 5.0 g/L, Na2HPO4 0.5 g/L, KCl 0.3 g/L, hemin 1.0 mg/L, fetal bovine serum (heat inactivated) 2% v/v, pH 6.5, and containing penicillin G sodium (106 units/L) and streptomycin sulfate (292 mg/L) at 27°C [101]. Free active Crithidias were observed 24 hours post inoculation. Parasites were maintained by subculture passage every 4 days; stable liquid nitrogen stocks were archived. Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (4′,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. Images of fixed Crithidia mellificae were obtained using both the Leica DM6000 microscope and a Zeiss LSM 510-M microscope equipped with both a 63× objective numerical aperture 1.4, and a 100× objective numerical aperture 1.4.For DNA purification, Crithidia mellificae (∼106 trypanosomes/mL culture medium) were pelleted by centrifugation (800×g for 6 min) and washed with PBS prior to DNA extraction. DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer's instructions. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).
PLoS One. 2011; 6(6): e20656.
Assessment of Arthropod Pathogen Microarray sensitivityIn order to estimate the sensitivity of the arthropod pathogen microarray (APM) two positive control samples were prepared in the presence and absence of pathogen-free honey bee RNA. A full-length (9,264 nucleotide) Drosophila C virus (DCV) clone was in vitro transcribed, serially diluted into honey bee RNA, reverse-transcribed, amplified, dye-labeled and hybridized to the APM as described above. Detection of at least 3 of the 8 unique DCV oligonucleotides and their reverse complements resulted in an estimated DCV detection level of 1.9×105 genome copies (1 pg DCV genomic RNA) in an A. mellifera RNA (1 µg) background. Similarly, detection of a BQCV genome segment (452 nt), corresponding to one array oligo and its reverse complement, diluted into either pathogen-free honey bee RNA (0.5 µg) or water indicated detection limits of 1.2×105 genome segment copies (30 fg BQCV RNA segment) and 1.2×104 genome segment copies (3 fg BQCV RNA segment) respectively.
PLoS One. 2011; 6(6): e20656.
Arthropod Pathogen Microarray design and synthesisDesign principles used for APM oligonucleotides (70 nt) were based on previous pan-viral microarrays using ArrayOligoSelector (AOS) [54]. Briefly, array oligonucleotides were selected for uniqueness against an insect nucleic acid background, for ∼50% GC content to maintain high complexity, and for cross-reactivity of highly-conserved nucleic acid features with evolutionarily related targets (<−50 kcal/mol predicted binding energy). Arthropod pathogen oligonucleotides (GEO GPL11490) were synthesized by Invitrogen, suspended at 40 pmol/µL in 3× SSC and 0.4 pmol/µL control oligo and printed on poly-L-lysine slides (Thermo) with silicon pins as previously described [100]. Each oligonucleotide and its reverse complement were printed twice for redundancy. Arrays were allowed to air-dry and stored and room temperature. Prior to use, oligonucelotides were cross-linked to slides via UV exposure (600 mJ), washed with 3× SSC/0.2% SDS and blocked using a methylpyrrolidone solution (335 mL 1-methyl-2-pyrrolidinone, 5.5 grams succinic anhydride, 15 mL 1 M sodium borate).
PLoS One. 2011; 6(6): e20656.
Honey bee sample preparationWe determined that analysis of five honey bees per sample was sufficient for our colony monitoring project. Arthropod pathogen microarray (APM) analysis of test samples revealed that combined analysis of 5 bees reproducibly detected most, if not all, of the pathogens detected from 10 or 15 independently analyzed bees from the same sample. In addition, we confirmed the consistency of APM results by performing multiple analyses of a single RNA sample. Based on our test results and practical sample handling considerations, we reasoned that repeated analysis of 5 bees from each colony over-time (115 bees per colony) was sufficient for this study.Honey bee samples, 5 bees per colony each time-point, were homogenized in 1 mL 50% TRIzol Reagent (Sigma) and 50% phosphate buffered saline (PBS, UCSF Cell Culture) solution in a 2 mL micro-centrifuge tube containing one sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch), for 4 minutes at 30 Hz. RNA was isolated according to TRIzol Reagent (Invitrogen) manufacturer's instructions. In brief, TRIzol reagent honey bee homogenate was combined with 0.1 ml chloroform and mixed by vortexing for 5 seconds, samples were incubated at room temperature for 5 minutes, prior to centrifugation for 10 minutes at 13,200× g in a table top centrifuge. Next, 700 µL of the aqueous phase was transferred to a new microfuge tube containing 490 µL isopropanol. Following mixing, the samples were incubated at −20°C for 20 minutes and then either centrifuged (13,200× g for 15 min) or further purified utilizing Zymo-III RNA columns according to manufacture's instructions (Zymo). RNA was extracted from five bees collected from the colony entrance for each of the time-course samples.
PLoS One. 2012; 7(5): e37361.
MiRNA Expression AnalysisRNA was isolated from MDA-MB-231(SA) and parental MDA-MB-231 cells using the mirVana miRNA Kit (Applied Biosystems). MiRNA microarray analyses were done by Agilent Technologies Inc. using the Agilent microarray platform v1, which contained probes for 455 human miRNAs. The data were normalized by variance stabilization [42].
J Clin Microbiol. 2011 December; 49(12): 4246–4251.
miRNA labeling and array hybridization. After RNA isolation from the samples, the miRCURY Hy3 power labeling kit (Exiqon, Vedbaek, Denmark) was used for the miRNA labeling of the two pooled total RNA groups according to the manufacturer's guidelines. Briefly, 1 μg of each sample was labeled using T4 RNA ligase in calf intestinal alkaline phosphatase (CIP) buffer and CIP (Exiqon). After stopping the labeling procedure, the Hy3-labeled samples were hybridized at 56°C overnight on the miRCURY LNA array (v.16.0) (Exiqon), containing probes for 1,223 human miRNAs, in a 12-bay hybridization system (NimbleGen Inc., Madison, WI). Following hybridization, the slides were washed several times using a wash buffer kit (Exiqon) and finally dried. Each miRNA spot was replicated four times on the same slide, and two microarray chips were used for each group.
J Clin Microbiol. 2011 December; 49(12): 4246–4251.
Microarray data analysis. The slides were scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). Scanned images then were imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Signal intensities for each spot were scanned and calculated by subtracting background levels. miRNAs with intensities of >50 were chosen for calculating the median normalization factor. Expressed miRNA data were normalized using median normalization. After normalization, obtained average values for each miRNA spot were used for statistics. Finally, hierarchical clustering was performed to show distinguishable miRNAs using MEV software (v4.6; TIGR [The institute for Genomic Research]).
