Comment[ArrayExpressAccession] E-GEOD-9950
Public Release Date 2008-06-01
Investigation Title Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci
Comment[Submitted Name] Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci
Experiment Description "It has been postulated that noncoding RNAs (ncRNAs) are involved in the post-transcriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. The complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, twenty two RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues. Keywords: tissue expression pattern 1. Experiment Design: 1.1. Type of experiment: The experiments described here aim to generate expression profiles from human and mouse prostate, kidney and liver tissue samples using a custom-designed cDNA microarray platform with 4,608 unique elements in replicate (9,216) enriched in gene fragments that map to intronic regions of known human genes. The platform consists of 3,355 probes of human cDNA fragments that map in the human genome sequence either to intronic regions of known genes (822), to unannotated regions of the human genome (241) or to known exons of RefSeq genes (2,292). We separately compared the expression profiles obtained from human and mouse samples to identify sequence conserved tissue-specific intronic transcripts. 1.2. Experimental factors: Tissues from liver, prostate and kidney from five adult male mice of the Balb/c strain were obtained and frozen in liquid nitrogen. Likewise, five non-tumor samples of human prostate, kidney or liver from our tumor bank were selected. All samples were obtained from patients who signed informed consent, and approval was received from the ethics committees of the hospitals. Total RNA was purified from tissues and treated with DNAse to reduce the levels of genomic DNA contaminant. Either human and or mouse samples from for each tissue from the five individuals were separately pooled using equal quantities of RNA. 1.3. The number of hybridizations performed in the experiment. RNA pools from each tissue sample was hybridized twice, performing a total of 18 hybridizations. 1.4. The type of reference used for the hybridizations, if any. All hybridizations included external mRNA spikes for quality control of the experiments (detailed below in section 2.5). Hybridizations were performed as single color experiments. 1.5. Hybridization design: Three aliquots of 15ug of the pool of total RNA from each tissue was labeled with Cy5-dUTP after reverse transcription (CyScribe first strand cDNA labeling kit, Amershan Biosciences) and hybridizations were carried out using all labeled-target in an Automate Slide Processor (Amershan Biosciences). Images were obtained using a Generation III Scanner (Amersham Biosciences). The human and mouse obtained expression data were independently analyzed using a highly stringent methodology, including different statistical approaches (see below for a detailed description). 1.6. Quality control steps taken: Integrity of total RNA was checked by electrophoresis using the Bio Sizing total RNA Nano assay in the 2100 Bioanalyzer (Agilent Technologies). Three replicates of each experiment were performed, as describe in the previous section. "
Date of Experiment
Term Source Name EFO
Term Source Version
Term Source File http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl
Person Last Name Louro Louro El-Jundi Nakaya Reis Verjovski-Almeida
Person First Name Rodrigo Rodrigo Tarik Helder Eduardo Sergio
Person Mid Initials I M
Person Email rolouro@iq.usp.br
Person Affiliation Instituto de Quamica da Universidade de Sao Paulo
Person Phone
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Person Address "Departamento de Bioquamica, Instituto de Quamica da Universidade de Sao Paulo, Av. professor Lineu Prestes, 748, Sao Paulo, Brazil"
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Protocol Name P-GSE9950-1 P-GSE9950-6 P-GSE9950-5 P-GSE9950-2 P-GSE9950-8 P-GSE9950-9 P-GSE9950-3 P-GSE9950-4 P-GSE9950-7
Protocol Description ID_REF = This column corresponds to the ID column of the platform
VALUE = Normalized signal intensities.
RAW_VALUE = Raw signal intensities (background subtracted) Images were obtained from each channel by laser scanning using a Generation III Scanner (Amersham Biosciences). Slides were scanned with the following parameters: excitation wave length: 633 nm (Cy5-labeled targets); emission filter: 675 nm (Cy5-labeled targets); PMT voltage: 700 volts (Cy5-labeled targets). "Labeled targets were ressuspended in 250 (l of 1 x hybridization buffer (25% formamide, 12.5% of proprietary Microarray Hybridization Buffer Version 2 from Amersham Biosciences cat. RPK0325) denatured for 2 min at 92 ºC and centrifuged at 13,000 rpm for 5 min. All slide processing steps (blocking, hybridization, washing) were carried out on an automated slide processor (ASP) from Amersham Biosciences. The slides were incubated for 16h at 42ºC and subsequently washed at room temperature in 1xSSC/0.2%SDS for 5min, 0.1xSSC/0.2%SDS for 5 min and in 0.1XSSC for 3 min. After the washing steps, slides were flushed with isopropanol and dried with an air flush at 42ºC." Total RNA purified using Trizol from frozen kidney tissues were treated with RNAse-free DNAse (RNeasy – Qiagen) for 15 minute to reduce the levels of genomic DNA contaminant. Total RNA purified using Trizol from frozen liver tissues were treated with RNAse-free DNAse (RNeasy – Qiagen) for 15 minute to reduce the levels of genomic DNA contaminant. Total RNA purified using Trizol from frozen prostate tissues were treated with RNAse-free DNAse (RNeasy – Qiagen) for 15 minute to reduce the levels of genomic DNA contaminant. Total RNA was purified from cell extracts using Trizol (Invitrogen). Total RNA was treated with of RNAse-free DNAse (RNeasy - Qiagen) for 15min to minimize genomic DNA contaminants. Purity of the isolated RNA was estimated by measuring the ratio A 260/A 280. Integrity of total RNA was checked by electrophoresis using the Bio Sizing total RNA Nano assay in the 2100 Bioanalyzer (Agilent Technologies). "Labeled targets for hybridizations were generated from total RNA in reverse transcription reactions using oligo dT as RT primer, following strictly the protocol accompanying the CyScribe First-Strand labeling kit (Amersham Biosciences, Piscataway, NJ). In short, 15 (g total RNA was combined with primers in a total volume of 11 (l, denatured at 70°C for 10 minutes, put on ice for 30 seconds, spin down and placed at room temperature for 10 minutes. Nine microliters of a premix solution was added containing 5X Superscript II reaction buffer (5X), 200 U Superscript II reverse transcriptase, 0.1 M DTT, dNTP mix (2 mM dATP, 2 mM dGTP, 2 mMdTTP ,1 mM dCTP) and 1 mM of Cy5-dCTP. After addition of the premix, the reaction was kept at room temperature for 10 minutes and then incubated at 42°C for 1.5 hr. RNA templates were removed by alkaline hydrolysis by adding 2 (l of 2.5 M sodium hydroxide and incubating the mixture at 37° C for 15 minutes. The labeling reaction was neutralized with 10 (l 2 M MOPS free acid and labeled targets were purified using 96-well Millipore Multiscreen filter plates as follows: 5 volumes of 5.3 M Guanidine-HCl: 150 mM KOAc were added to labeling reactions. The mixture was applied on the plate and washed 4X with 80% EtOH by centrifugation at 3500 rpm for 5 min. Residual EtOH was spin out by an additional centrifugation at 3500 rpm for 5 minutes. Labeled targets were eluted in 50 (l 10mM Tris pH 8.5, by spinning at 3000 rpm for 5 minutes, dried on a SpeedVac and kept at -20°C, protected from light until use." "We employed in our analyses the Artifact-Removed density value (ARM) calculated for each spot by ArrayVision. The ARM value represents the average of all the pixels remaining in the spot, after first removing pixels with density values that exceed four median absolute deviations (MADs) from the median. The ARM density for each spot was subtracted by the median background surrounding the spot (distance of 2 pixels with 3 widths of pixels).The raw data of each spot was compared to the value of hibridization background using the Lucidea Microarray Scorecard to determine which spots have a signal above or below the detection limit of each hybridization, where the background was given by the signal measured on a negative control (plant cDNA). The 3,686 unique probes are duplicated in each slide (Left and Right sides of the slide), and each transcript was considered as expressed in one side of the slide if its probe intensity was equal to or higher than the mean intensity of the Negative Controls plus three standard deviation in the same sub array. The gene was considered detected in the slide if it was positive on both sides (Left and Right) and was considered as expressed in the tissue if it was detected in all three slides. It was considered as detected in each species if it was detected in at least one tissue. The intensities of all spots of each slide were normalized among the different experiments using the 40% trimmed average intensity of a set of six different housekeeping genes (spotted at 264 different places in each slide) as reference."
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Protocol Type bioassay_data_transformation image_aquisition hybridization specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction labeling feature_extraction
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Publication Title Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci.
Publication Author List "Louro R, El-Jundi T, Nakaya HI, Reis EM, Verjovski-Almeida S"
PubMed ID 18495418
Publication DOI 10.1016/j.ygeno.2008.03.013
Publication Status
Publication Status Term Source REF
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Comment[SecondaryAccession] GSE9950
Comment[GEOLastUpdateDate] 2008-08-31
Comment[GEOReleaseDate] 2008-05-31
Comment[ArrayExpressSubmissionDate] 2007-12-18
SDRF File E-GEOD-9950.sdrf.txt