|
| Status |
Public on Jan 15, 2016 |
| Title |
Female wild-type littermate control-intact 4180 |
| Sample type |
RNA |
| |
|
| Source name |
LVS0734
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 N background
genotype: wild-type littermate
gender: Female
tissue: Heart-left ventricle
Stage: Control-intact
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1. 20-30 mg mid-ventricular section (previously stored at -80 C) has been placed into 600 µL RLT buffer in a tissue disintegration tube (Lysing Matrix D, MP Biomedicals, Cat. Nr. 6913-100 )
2. Disintegration was performed using RiboLyser for 40s at maximal power “6". Samples were stored on ice during the transfers.
3. Lysing matrixtubes were centrifuged for 1 min at 10 000 x g at room temperature and supernatant transferred into a new tube.
4. Lysing matrix was washed using additional 290 µL RNase free water, centrifuged for 1 min at 10 000x g supernatants were joined.
5. Proteinase K (provided in the RNeasy Fibrous tissue Mini kit) was added (10 µL per sample) and mixed thoroughly.
6. Extraction continued with the RNeasy FibrousTissue Mini Kit; Qiagen; Cat Nr: 74704 according to manufacturer's manual
|
| Label |
Cy3
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 1 µg of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double stranded cDNA in a reverse transcription reaction. Subsequently the sample was converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) in an in vitro transcription reaction according to the manufacturer’s protocol (Agilent).
|
| |
|
| Hybridization protocol |
Purified and labeled cRNA (28 pmol of Cy3 labeled cRNA) was hybridised on Agilent Mouse Whole Genome 4x44k array (design 014868) followed by (manual) washing, according to the manufacturer’s procedures.
|
| Scan protocol |
To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with surescan High-Resolution Technology and probe signals were quantified using Agilent’s Feature Extraction software (version 9.5.1.1).
|
| Description |
Mid-ventricular 20-30 mg ring of cardiac tissue; homogenized using desintegrator with Lysing Matrix D beads
|
| Data processing |
The dataset contains the Agilent processed signal values (i.e., feature gProcessedSignal from Agilent Feature Extraction v9.5.1.1) after an additional quantile normalization. Intensities were base 2 log-transformed.
|
| |
|
| Submission date |
Apr 26, 2012 |
| Last update date |
Jan 15, 2016 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE37597 |
Cardiomyocyte-specific Nitric Oxide Synthase 3 Overexpression in Pressure-Overloaded Left Ventricle. |
|
