|
| Status |
Public on Mar 31, 2014 |
| Title |
Liver-1 |
| Sample type |
RNA |
| |
|
| Source name |
Liver. Samples from 3 different pig were extracted and than pooled because pigs were not inbred.
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: Liver
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. Quality of small RNA of pooled samples from the same tissue was tested on Agilent Bioanalizer 2100 using the RNA small LabChip.
|
| Label |
Cy3
|
| Label protocol |
The small RNA was not labelled when it was used to probe microarray. RNA-primed Array-based Klenow Extension (RAKE) is based on the ability of a RNA molecule to function as a primer for Klenow polymerase extension when fully base-paired with a microarray probe. As we known the exact 3’ end of the miRNA by previously performed tailed microarray experiments, we designed a microarray with a specific probe for each 3' end identified and a background probe. This microarray design was avoided for known miRNAs.
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| |
|
| Hybridization protocol |
Microarrays were probed with 800-900 ng of the small RNA from each tissue for 20 hours at 37° C in a static hybridization oven (SSPE 6X; BSA 8 mg/ml; 800 - 900 ng of small RNAs and spike-in). First, microarrays were pre-hybridized for 2 hours at 37° C with the following pre-hybridization water solution: SSPE 6X and BSA 8 mg/ml. After the retrotranscribed small RNAs hybridization, microarrays were washed with the following stringent washing solutions: 1 minute at room temperature with 6x SSPET (SSPE added with 0.05% of Tween-20); 1 minute at room temperature with 3x SSPET; 1 minute at room temperature with PBS 2X; 1 minute at room temperature with Buffer 2, 1X (the buffer for the klenow enzyme). After the washing step it was performed the RAKE reaction at 36.5° C incubating the microarray for 1 hour and 30 minutes with the following solution: Buffer 2 1X; Biotin-14-dATP (Invitrogen) 16 μM; Klenow Fragment (3´→5´ exo–) (NEB) 0.25 U/μl. Than microarray was washed two times with Buffer 2 1X and than incubated with the biotin blocking solution (PBS 2X; Tween-20 0.1% and BSA 10 mg/ml) for one hour at room temperature. Extended miRNAs (primers) were labeled incubating the microarray with the Dye labeling solution (PBS 2X; Tween-20 0.1% ; BSA 10 mg/ml and 1.6 ng of Cy3-streptavidin; Amersham) for one hour at room temperature. Microarray was rinsed with PBST (PBS 2X added with Tween-20 0.1%) for one minute at room temperature and with PBS 2X for one minute at room temperature.
|
| Scan protocol |
Microarrays were scanned with the VersArray scanner (Biorad) (6 μm resolution).
|
| Description |
Tissue was extracted from three different pig (12 month old) and small RNA was pooled.
RAKE using hybridized miRNA as primer. Incorporated biotin dATP was labelled with Cy3-streptavidin
|
| Data processing |
The RAKE experimental setup associates fluorescence levels to specific and backrground probes. Before spike-in based miRNA quantization inter-arrays fluorescence was normalized using cyclic loess method . This is an algorithm that allows good grade of normalization in miRNA gene expression experiments as discussed by Wu (Comparison of normalization methods for CodeLink Bioarray data. BMC Bioinformatics) exhibiting the best improvement in the reduction of variability and yield the highest number of significant differentially expressed miRNAs. After inter-arrays normalization intensity fluorescence of specific probe for the miRNA was subtracted of the corresponding background fluorescence and than used to extrapolate concentration basing the spike-in derived curve.
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| |
|
| Submission date |
Mar 23, 2011 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
[email protected]
|
| 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 |
GPL13322 |
| Series (2) |
| GSE28140 |
Evaluation of miRNA expression in 14 different tissues by the RAKE technology |
| GSE28637 |
Discovering, evolution, biogenesis, expression and target prediction of porcine micro-RNAs: new regulatory gene expression network in different tissues. |
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