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| Status |
Public on Dec 29, 2020 |
| Title |
cochlear_tissue_Cx30-/-_mice_rep2 [mRNA] |
| Sample type |
RNA |
| |
|
| Source name |
cochlear_tissue_Cx30-/-_mice_rep2
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Cx30-/-
tissue: cochlear tissue
age: Post-natal day 5
|
| Growth protocol |
A combination of three different primers was used to identify the Cx30 interruption due to LacZ insertion in Cx30-/-mice: Cx30F: 5’-GGTACCTTCTACTAATTAGCTTGG-3’, Cx30R: 5’-AGGTGGTACCCATTGTAGAGGAAG-3’, Cx30lacZ: 5’-AGCGAGTAACAACCCGTCGGATTC-3’. Mice were maintained on a pure C57BL/6N background. All experimental protocols were carried out in accordance with the relevant guidelines and regulations and approved by the Ethical Committee of Padua University and by the Italian Ministry of Health.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA including miRNAs was extracted from mice cochlear tissue (at post-natal day 5) using Absolutely RNA Miniprep Kit (cat number #400800, Agilent Technologies, Santa Clara, CA, USA), according to manufacturer’s instructions. To detect possible contaminants, the ratios of absorbance were assessed at 260/280 nm and 260/230 nm using the NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific). The quality of the RNA was evaluated with Agilent 2100 Bioanalyzer microfluidic electrophoresis platform, using the Small RNA assay for miRNAs, following the manufacturer's specifications. All samples passed the quality evaluation and were processed for profiling experiments.
|
| Label |
Cy3
|
| Label protocol |
Messenger RNA was cyanine-3 (Cy3) labeled starting from 100 ng of total RNA of each sample using the One-Color Microarray-Based Gene Expression Analysis - Low Input Quick Amp Labeling kit protocol (Version 6.9.1, December 2015), according to the manufacturer's instructions. This kit generates fluorescent cRNA from cDNA, by means of T7 RNA Polymerase Blend that simultaneously amplifies target material and incorporates Cy-3. Subsequently, labeled amplified cRNA purification was carried out using RNeasy mini kit (Qiagen) before to be quantified by Nanodrop ND-1000 spectrophotometer (ThermoFisher Scientific) to determine yield and specific activity.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA was fragmented in a reaction volume of 250 ml containing fragmentation buffer and blocking agent, following the manufacturer's instructions. On completion of fragmentation step, the hybridization buffer was added to the fragmentation mixture and 40 ul of each sample were then hybridized to SurePrint G3 Mouse GE v2 microarray (8X60K, Design 074809). The assembled slide cassette was placed into the hybridization oven and hybridized for 17 hours at 65°C in a rotating Agilent hybridization oven. Disassembling washing step was carried out using the gene expression wash buffer 1 at room temperature, followed by the 1st wash using gene expression wash buffer 1 for 1 minute at room temperature and the 2nd wash using gene expression wash buffer 2 for 1 minute at 37°C.
|
| Scan protocol |
The microarrays were scanned at 3 μm resolution using a SureScan Microarray Scanner System (Agilent Technologies). The scanned images were analyzed with Feature Extraction Software v. 11.5.1.1 (Agilent Technologies) for acquisition, data extraction and quality control analysis. After the evaluation of quality control parameters for each scanned microarray images, the raw data of all samples were further analyzed.
|
| Description |
SAMPLE 3_D1
Gene expression at P5 in cochlear tissue of Cx30-/- mice.
|
| Data processing |
GeneSpring GX v.14.5 software (Agilent Technologies) was used to carry out data analysis. Fluorescence signal values were thresholded to 1, log2 transformed, normalized to the 75th percentile, baselined to the median of all samples, and quality-filtered on flags to include any probe detected in 100% of biological replicates in at least one out of two of tested experimental conditions. In order to identify differentially expressed genes (DEGs) between knock-out relative to wild-type, a moderate T-test was performed, followed by Westfall-Young multiple testing correction procedure, and adjusted p-values < 0.05 were used as criteria for defining a set of deregulated candidate genes for further exploration.
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| |
|
| Submission date |
May 28, 2020 |
| Last update date |
Dec 29, 2020 |
| Contact name |
Sebastiano Cavallaro |
| E-mail(s) |
[email protected]
|
| Organization name |
IRIB-CNR
|
| Street address |
Via Paolo Gaifami, 18
|
| City |
Catania |
| ZIP/Postal code |
95126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL21163 |
| Series (2) |
| GSE151367 |
MicroRNA and mRNA integrated transcriptional analysis in a Cx30-/- mouse model of non-syndromic hearing loss and deafness [mRNA] |
| GSE151369 |
MicroRNA and mRNA integrated transcriptional analysis in a Cx30-/- mouse model of non-syndromic hearing loss and deafness |
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