|
| Status |
Public on Sep 15, 2020 |
| Title |
25EDLEt |
| Sample type |
RNA |
| |
|
| Source name |
EDLTransgenic25
|
| Organism |
Mus musculus |
| Characteristics |
tissue: EDL muscle
gender: male
mutation: Ile4898Thr
|
| Treatment protocol |
no treatment
|
| Growth protocol |
RyR1 I4895T knock-in mice on a 129S2/SvPasCrl background were kindly provided by the S. Hamilton laboratory, Texas, USA. These founder mice were rederived using C57BL/6J for breeding. This resulted in a new sub-line with a mixed genetic background (approximately 50% 129S2/SvPasCrl and 50% C57BL/6J after rederivation). Since homozygotes have a perinatal lethal phenotype (Zvaritch et al. 2007), heterozygotes (HET) were crossed with wild type (WT) littermates or C57BL/6J mice (Charles River) to maintain the colony. At least five such crosses had occurred before the experiments. Ear snips from adult mice and tail snips from sacrificed neonates were retained for genotyping. DNA was extracted from tissue samples using the Extract-N-Amp kit (Sigma) and PCR was used to amplify the fragment covering the mutation site. Forward and reverse primers were 5’-GGTCTTCCTGTCTCAATAACCCGATCTAGAAAC-3’ and 5’-GATGGAGAAACCAAAGCTCAGAGAGACCAC-3’ respectively. Since the inserted mutation site also contains an Age I target sequence, only samples containing the mutated allele undergo Age I digestion.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from biopsies using TRIzol (Life Technologies) following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA was prepared from 1 μg of RNA using One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) (Agilent Technologies) according to the manufacturer's instructions, and RNeasy column purification was performed (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
Cy3-labeled cRNA (1.65 μg; specific activity of >9.0 pmol Cy3/μg of cRNA) was fragmented at 60 °C for 30 min in a reaction volume of 55 μl containing 1× Agilent fragmentation buffer and 2× Agilent blocking agent, following the manufacturer's instructions. On completion of the fragmentation reaction, 55 μl of 2× GEx Hybridization buffer HI-RPM was added to the fragmentation mixture, and mixtures were hybridized to Agilent Whole-Human Genome Oligo Microarrays (G4845A) for 17 h at 65 °C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 1 min at 37 °C with GE Wash buffer 2 (Agilent Technologies) and then dried
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using the one-color scan setting for 1 × 44 k array slides (scan area of 61 × 21.6 mm; scan resolution of 5 μm; dye channel set to green; green PMT set to 100%).
|
| Data processing |
Scanned images were analyzed with Feature Extraction Software 12.0.0.7 (Agilent Technologies) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_20070820 ) to obtain background-subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Signal intensities were normalized using quantile normalization for GE .
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| |
|
| Submission date |
Sep 14, 2017 |
| Last update date |
Sep 15, 2020 |
| 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 |
GPL7202 |
| Series (2) |
| GSE103853 |
Inositol Trisphosphate Receptor Mediated Ca2+ Signalling Stimulates Mitochondrial Function and Gene Expression in Core Myopathy Patients [Mouse arrays] |
| GSE103855 |
Inositol Trisphosphate Receptor Mediated Ca2+ Signalling Stimulates Mitochondrial Function and Gene Expression in Core Myopathy Patients |
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