|
| Status |
Public on Mar 31, 2014 |
| Title |
Kidney-2 |
| Sample type |
RNA |
| |
|
| Source name |
Kidney. Samples from 3 different pig were extracted and than pooled because pigs were not inbred.
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: Kidney
age: 12 month old
breed: vietnamite
|
| Treatment protocol |
After the sedation, pigs were sacrificed and portions of the described organs were took and maintained in the RNALater solution (Ambion), excluding blood that was collected in the BD Vacutainer CPT cell preparation tubes, and processed according to manufacturer's directions to recover white blood cells..
|
| Growth protocol |
Pigs were maintained at normal food regime
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA and miRNAs were extracted independently from each tissue sample by TRIzol reagent (Invitrogen) in association with PureLink miRNA isolation kit (Invitrogen). Briefly, approximately 200 mg of tissue was homogenized in 3.5 ml of TRIzol reagent (Invitrogen) using a tissue homogenizer (IKA Werke). After chloroform addition and centrifugation colorless upper aqueous phase containing RNAs was added with 96-100% ethanol to obtain a final concentration of 35% of ethanol and charged to PureLink membrane (Invitrogen) to separate total RNA and small RNAs from the same sample following the manufacturer manual. Total RNA and small RNAs were quantized using Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific). Samples derived from the same tissues were pooled adding the same quantity from the three pigs used. “Pool tissues” were performed using 1 ug of total RNA from each sample while for small RNAs it was used 1.5 ug from each sample. Quality of total RNA of pooled samples was tested on Agilent Bioanalizer 2100 using RNA 6000 Nano LabChip.
|
| Label |
Cy3
|
| Label protocol |
1 μg of total RNA was amplified with biotin dUTP and after hybridization the Cy3-streptavidin was added. Amplification was performed with the Ambion MessageAmp™ II aRNA Amplification kit (Ambion). The procedure includes reverse transcription with an oligo-dT primer bearing a T7 promotor using reverse transcriptase to produce the first-strand cDNA. After second-strand synthesis and cleanup, the cDNA becomes a template for the in vitro transcription technology to generate antisense RNA (aRNA) copies of each mRNA molecule. Biotinilated UTPs were incorporated into the aRNA during the in vitro transcription reaction. Following purification 18 μg of aRNA was fragmented using the Ambion Fragmentetion Kit (Ambion). aRNAs and fragmented aRNAs were tested on Agilent Bioanalizer 2100 using RNA 6000 Nano LabChip. All aRNAs presented length from 300 and 4,000 nucleotides while fragmented aRNAs from 50 and 250 nucleotides.
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| |
|
| Hybridization protocol |
Fragmented aRNA was hybridized to pre-hybridized 90K Combimatrix microarrays. Pre-hybridization was performed for 2 hours at 42° C with the following solution: 1X Hyb solution stock prepared as suggested by Combimatrix; 5X Denhardt’s solution; Salmon sperm DNA 100 ng/μl; SDS 0.05%. Hybridizations were performed with 4.8 μg of fragmented aRNA at 42° C for 18 hours mixing the hybridization solution (1X Hyb solution stock prepared as suggested by Combimatrix; 25% of DI Formammide; Salmon sperm DNA 100 ng/μl; SDS 0.04% and 4.8 ug of fragmented aRNA). After hybridization microarrays were washed with: 6X SSPET (SSPE added with 0.05% of Tween-20) preheated at 42° C for 5 min.; 3X SSPET for 1 min. at room temperature; 0.5X SSPET for 1 min. at room temperature; PBST for 1 min. at room temperature; The array chamber was than filled with Biotin Blocking solution and incubated at room temperature for 1 hour. Labeling was performed incubating the microarray with the Dye labeling solution (Cy3 streptavidin in biotin blocking solution) for 1 hour at room temperature. After we performed these wash steps: PBST for 1 min. at room temperature two times; PBS for 1 min. at room temperature.
|
| Scan protocol |
Microarrays were scanned at 3 μm resolution with the VersArray ChiprRaderTM (BioRad)
|
| Description |
Tissue was extracted from three different pig (12 month old) and total RNA was pooled.
|
| Data processing |
Before performing all analysis replicated probes (same probe sequence) were averaged (FG Median values were used). Raw data were normalized with the quantile method. After data normalization intensity fluorescence of genes presenting the expression lower than the average of median of all negative controls plus 1 standard deviation (see Filter Table) was set as NA. We used the negative controls present on the array to calculate background value used in the filter table. Genes presenting NA in more than 6 experiments were excluded from the analysis (see Analysis Table).
R based quantile normalization.
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| |
|
| Submission date |
Mar 09, 2011 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
stefano.cagnin@unipd.it
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13259 |
| Series (2) |
| GSE27853 |
Messenger RNA transcript expression in the pig (S. scrofa) |
| GSE28637 |
Discovering, evolution, biogenesis, expression and target prediction of porcine micro-RNAs: new regulatory gene expression network in different tissues. |
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