|
| Status |
Public on Dec 13, 2014 |
| Title |
Subject6_day2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Muscle cells_differentiating_2 days
|
| Organism |
Homo sapiens |
| Characteristics |
subject id: subject 6
tissue: vastus lateralis muscle
cell type: Human skeletal muscle cells
time point: 2 days after induction of differentiation
|
| Growth protocol |
Human skeletal muscle cells (extracted from vastus lateralis muscle) was cultured in Dulbecco's Modified Eagle Medium (DMEM):Nutrient Mixture F-12 supplemented with 20% FBS, 1% fungizone and 1% Penicillin-Streptomycin. Cultures were subcultured with trypsin before reaching 70% confluence. Differentiation of myoblasts into myotubes were induced by switching to differentiation medium (DMEM (low glucose) supplemented with 2% FBS, 1% PeSt and 1% Fungizone) when cells were 80% confluent. Cultures were incubated at 37°C in 7.5% CO2 humidified chambers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA by TRIzol (Invitrogen). Modifications of manufacturer's protocol include addition of 0.75 ml isopropanol (compared to 0.5 ml) containing glycogen with 20 minutes incubation at -20°C to precipitate RNA.
|
| Label |
Hy3
|
| Label protocol |
300 ng of total RNA from samples and a reference pool was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
Mixed pool of total RNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Total RNA from pooled samples from channel1 input.
|
| Growth protocol |
Human skeletal muscle cells (extracted from vastus lateralis muscle) was cultured in Dulbecco's Modified Eagle Medium (DMEM):Nutrient Mixture F-12 supplemented with 20% FBS, 1% fungizone and 1% Penicillin-Streptomycin. Cultures were subcultured with trypsin before reaching 70% confluence. Differentiation of myoblasts into myotubes were induced by switching to differentiation medium (DMEM (low glucose) supplemented with 2% FBS, 1% PeSt and 1% Fungizone) when cells were 80% confluent. Cultures were incubated at 37°C in 7.5% CO2 humidified chambers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA by TRIzol (Invitrogen). Modifications of manufacturer's protocol include addition of 0.75 ml isopropanol (compared to 0.5 ml) containing glycogen with 20 minutes incubation at -20°C to precipitate RNA.
|
| Label |
Hy5
|
| Label protocol |
300 ng of total RNA from samples and a reference pool was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
Hybridization to miRCURY™ LNA array version 11.0 arrays (Exiqon, Denmark) was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria).
|
| Scan protocol |
Microarrays were stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. Slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA).
|
| Description |
Sample 12
|
| Data processing |
Image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). Quantified signals were background corrected (Normexp with offset value 10) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.
Data repressents normalized log2 ratio (Hy3/Hy5) representing test/reference. When calling of a particular miRNA failed on an array this is indicated by 'null'. In the expression matrix, all capture probes with both Hy3 and Hy5 signals lower than 1.5x of the median signal intensity of the given slide, or which are annotated null, are excluded as indicated by 'null'.
|
| |
|
| Submission date |
Dec 16, 2013 |
| Last update date |
Dec 13, 2014 |
| Contact name |
Anna Krook |
| E-mail(s) |
anna.krook@ki.se
|
| Organization name |
Karolinska Institutet
|
| Street address |
von Eulers väg 4a
|
| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL15829 |
| Series (2) |
| GSE53383 |
Dynamic time course miRNA profiling of human skeletal muscle cell differentiation. |
| GSE53384 |
miRNA-transcriptome expression pattern during differentiation in human skeletal muscle cells |
|
