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| Status |
Public on Jun 01, 2008 |
| Title |
M-Liver_rep3_R |
| Sample type |
RNA |
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| Source name |
Liver pool
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| Organism |
Mus musculus |
| Characteristics |
Tissues from liver, prostate and kidney from five adult male mice of the Balb/c strain were obtained and frozen in liquid nitrogen.
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| Treatment protocol |
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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
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).
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| Label |
Cy5-dCTP
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| Label protocol |
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.
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| Hybridization protocol |
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.
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| Scan protocol |
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).
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| Description |
Hybridization included external mRNA spikes for quality control of the experiment (Lucidea Microarray ScoreCard. Raw images were analyzed using the ArrayVision software (Version 8.0, Imaging Research Inc.). An array template (or grid) was first automatically aligned to locate the position of each spot in the array, and subsequently manually adjusted to obtain the best possible alignment.
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| Data processing |
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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| Submission date |
Dec 18, 2007 |
| Last update date |
Dec 20, 2007 |
| Contact name |
Eduardo M Reis |
| E-mail(s) |
[email protected]
|
| Organization name |
Instituto de Química da Universidade de São Paulo
|
| Department |
Departamento de Bioquímica
|
| Street address |
Av. professor Lineu Prestes, 748
|
| City |
São Paulo |
| ZIP/Postal code |
05508-900 |
| Country |
Brazil |
| |
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| Platform ID |
GPL3985 |
| Series (1) |
| GSE9950 |
Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci |
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