MiRNA Microarray Profiling with Three Different Total RNA Prep Methods
ABSTRACT: We performed miRNA microarray profiling on samples prepared from two different cell lines by three widely-used total RNA sample prep methods. miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. For total RNA preps, frozen cell pellets were resuspended in phosphate buffered saline and divided into equal aliquots of 5x106 (HeLa) or 1x107 (ZR-75-1) cells and refrozen. Individual aliquots were subsequently thawed just before use.
Project description:We measured the expression of 470 human miRNAs in nine human tissues using Agilent DNA microarrays. miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. The labeling and hybridizations of the nine human tissues were done 4-5 times using the same starting RNA sample for each tissue.
Project description:To identify microRNAs potentially involved in melanomagenesis we compared microRNA transcription profiles between melanoma cell lines and cultured melanocytes. miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. The labeling and hybridizations of the nine human tissues were done 4-5 times using the same starting RNA sample for each tissue. Total RNA was extracted from 51 melanoma cells and 2 pools of melanocytes using the QIAGEN miRNA kits.
Project description:In order to infer the inverse expression relationships between microRNAs and mRNAs during HCV infection, we profiled both microRNA and mRNA expressions in HCV positive (HCV+) and HCV negative (HCV-) human liver biopsy tissue samples. This series includes all microRNA profiles. This series includes microRNA profiles of 36 samples, 24 HCV positives and 12 HCV negatives.
Project description:This SuperSeries is composed of the following subset Series: GSE11806: miRNA Microarray Profiling of Nine Different Normal Human Tissues GSE11840: miRNA Microarray Profiling with Three Different Total RNA Prep Methods Refer to individual Series
Project description:miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p. Organism used: Homo sapiens. * Slides: Human miRNA V2 8x15k Arrays AMADID: 19118. * Starting material: 18 slides with formalin fixed Paraffin embedded sections-20 μm. * RNA Samples used: 225-94-DUP, 225-94-E, 727-07-DUP, 727-07 -E. * Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit. * Labeling Method: Ligation of one cyanine3-pCp molecule to the 3' end of RNA molecule with greater than 90% efficiency that generates fluorescent miRNA. * Total RNA and cRNA Purification Kit: Biorad MicroBio Spin 6 columns. * Hybridization Kit: Agilent miRNA Hybridization kit. Hybridization protocol See Agilent Technologies website for miRNA microarray hybridization and wash protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50500 Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2505C) at 100% PMT with 10% for lower intensity (XDR Scanning).Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol Data processing Feature extracted data was analyzed using GeneSpring GX v 10.0.2 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended Percentile shift normalization (50th percentile) . Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. High expressed miRNA's were clustered using gene tree to identify significant gene expression patterns.
Project description:A database of apparently benign copy number variants (bCNVs) detected by a Spectral Genomics Inc./PerkinElmer BAC array platform has been maintained through the University of Utah Comparative Genomic Hybridization laboratory since 2005. The target population for this database represents 1275 patients with abnormal phenotypes, primarily children referred for developmental delay and mental retardation. These bCNVs are independent of any identified copy number abnormality detected. The most common 35 bCNVs observed and their frequencies are reported here, and a subset of ten of the patients studied was evaluated on a new oligonucleotide CNV array set designed by Agilent Technologies. There was a 76% concordance of calls detected by both array platforms in the same patients; the discordant regions may be due to differences in reference DNAs, or false positive/negative results on either array. The higher resolution of the Agilent oligonucleotide array compared to the BAC array allowed for further characterization to determine precise breakpoints of the observed CNVs, in addition to documenting additional CNVs of smaller sizes. As expected, observed CNVs and their frequencies were generally consistent with those of other previously published and available databases, including the Database of Genomic Variants (http://projects.tcag.ca/variation/). The availability of these data should assist other clinical laboratories in the evaluation of CNVs of unknown clinical significance. The Cytogenetics Laboratory at the University of Utah has been using aCGH to evaluate DNA derived from blood of patients with developmental delay, mental retardation and “syndromic” or dysmorphic phenotypes since 2005. From this database, we were able to determine which clones represented sequences which were frequently deleted or duplicated in our patient population. We identified our most common CNV regions and then selected DNAs from ten patients who exhibited copy number changes of those common regions for further study with the Agilent CNV microarray set. Normal male or female DNA from Promega was used as a sex-mismatched control. In addition to the ten clinical specimens, two self-self experiments for two patient samples were analyzed to estimate a false positive rate and to fine tune the parameters used for data analysis. A replicate hybridization was performed on one patient to determine reproducibility.
Project description:Despite considerable excitement over the potential functional significance of copy number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at ~1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1,020 of 1,153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of common human CNVs may be smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with inter-individual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously underappreciated complexity. The DNA samples are a panel of 30 Hapmap samples, and a female sample NA15510 of undetermined geographic origin, which has been well characterized by fosmid-end sequencing (Tuzun et al. 2005, Nature Genetics 10, p1038). The DNA for this set of 16 female and 15 male samples are all supplied by the Coriell Cell Repository, and is comprised of samples from four populations: 10 unrelated Yoruban, 10 unrelated European-American individuals from Utah (CEPH), 5 unrelated Chinese, and 5 unrelated Japanese. The reference sample, NA10851 is also a male CEPH also from the HapMap Sample set (Corriel Cell Repository). Each of these samples was hybridized in pairs with the reversed labeling polarities. Additionally, 3 self-self control hybridizations were carried out for the reference sample, NA10851, one on each hybridization date. In the Title for each of the samples, each hybridization session is indicated by the letters A, B or C.
Project description:Genetic variation amongst individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single-nucleotide changes. In this manuscript we explore variation on an intermediate scale-particularly insertions, deletions, and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number among individuals. Sequencing of a subset of structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence-map of human structural variation-an important standard for genotyping platforms and a prelude to future individual genome sequencing projects. Keywords: comparative genomic hybridization The DNA samples are a panel of 8 Hapmap samples, described by E. Eichler et al. (2007, Nature 447, 161-165). This set of 7 female, and one male samples are from from the Coriell Cell Repository, and is comprised of samples from four populations: four Yoruban, two CEPH, one Chinese, and one Japanese. The reference sample, NA15510, is female and also from the Corriel Cell Repository. This sample has been extensively characterized, (for example in Tuzan et al. 2005, Nature Genetics 10, p1038) and has been recommended for use in CNV detection programs to allow meaningful comparison of data between studies (discussed in Scherer, et al. 2007, Nature Genetics Supplement 39: S7-S15). Each of these samples was hybridized in pairs with the reversed labeling polarities. Additionally, 3 self-self control hybridizations were carried out for the reference sample, NA15510, one on each hybridization date.
Project description:We used microarrays to identify genes regulated by HCF-1 in a daf-16-dependent manner. Samples from young adult worms were hybridized to Agilent microarrays to identify genes differentially expressed in hcf-1(pk924)/daf-16(mgDf47);hcf-1(pk924) and compared to hcf-1(pk924)/N2 arrays.
Project description:Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine neoplasias of neural crest origin. They can be part of several syndromes, and their mRNA profile is dependent on genetic background, but questions related to clinical behavior or even main location remain unanswered. MicroRNAs are key modulators of target genes through translational repression, mRNA degradation, or both, and therefore they could resolve some of these issues. To determine the role microRNAs play in tumorigenesis and progression of PCC/PGL, as well as to identify microRNA biomarkers specifically related to different PCC/PGL genetic classes known so far, we characterized microRNA profiles in a large series of frozen tumors with germline mutations in SDHD, SDHB, VHL, RET, NF1, and TMEM127 genes through microarray analysis. We identified microRNA signatures specific to, as well as common among, the genetic classes of PCC/PGLs, and the best candidate microRNAs (miR-122, miR-126*, miR-129*, miR-133b, miR-137, miR-183, miR-210, miR-382, miR-488, miR-885-5p, and miR-96) were validated in an independent series of formalin-fixed paraffin-embedded PCC/PGL samples by qRT-PCR. MicroRNA-137, -96/183, and -143/145 expression in PCC/PGLs correlated inversely with the differentiation status of tumor cells. MicroRNA-210, -382, and -380 could modulate pseudohypoxic cellular response in VHL-deficient PCC/PGL. MicroRNA-193b, -365, and -424 were commonly downregulated among all genetic classes, suggesting their involvement in cell cycle control and differentiation. Herein, we demonstrate that PCC/PGLs have different microRNA profiles according to the underlying primary mutation, suggesting they could be used as specific biomarkers and add information on the etiology of these tumors. For this study, we employed the Agilent-019118 Human miRNA Microarray 2.0 G4470B (with Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit, Cat # 5190-0408) to perform microRNA expression profiling on a large series of 54 fresh-frozen tissue samples, including a total of 48 genetically characterized pheochromocytomas (n=37) and paragangliomas (n=11) (8 SDHB-related tumors, 4 SDHD-related tumors, 14 Ret-related tumors, 12 VHL-related tumors, 4 NF1-related tumors, 3 TMEM127-related tumors, and 3 familial pheochromocytoma (FPCC) samples), and 6 normal adrenal medulla. Normalization of array data was performed applying the robust multiarray average (RMA) method using the AgiMicroRna package in Bioconductor. RMA normalization, by default, merges replicates of each probe and produces an Eset with a single value for each microRNA.