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| Status |
Public on Dec 20, 2019 |
| Title |
C2C12 myoblasts transfected with miR-27a-3p rep4 |
| Sample type |
RNA |
| |
|
| Source name |
C2C12 myoblasts transfected with miR-27a-3p
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
cell type: myoblasts
transfected with: miR-27a-3p mimic
|
| Treatment protocol |
C2C12 myoblasts were cultured in Dulbecco's Modified Eagle Medium with high glucose (Life Technologies) + 10% Fetal Bovine Serum (Life Technologies) in 10 cm dishes and split every 2 or 3 days before they reached 70% of confluence. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer’s instructions with 150 nM of mirVana miRNA mimics or scrambled sequence, purchased from Life Technologies.
|
| Growth protocol |
C2C12 cells were cultured using standard protocols provided by the ATCC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from transfected cells was isolated using the standard phenol-chloroform extraction with TRIzol Reagent (Thermo Fisher Scientific) followed by ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
RNA obtained from cells was amplified and labeled starting from 150 ng using the Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) as described by the proprietary protocol. Labeled cRNA was purified with miRNeasy Mini Kit (Qiagen) and quantified using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). On average, cRNA yield was about 10 µg and the specific activity was of 35 pmol Cy3 per µg cRNA.
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| |
|
| Hybridization protocol |
800 ng of labeled sample target were mixed with 5 µl of 10X Blocking Agent (Agilent Technologies) and water to a final volume of 25 µl. Samples were denatured at 95°C for 2 min. and added to 25 µl of 2X GEx Hybridization Buffer HI-RPM (Agilent Technologies). 40 µl of mix was dispensed onto the array. Slides were loaded into the Agilent SureHyb chambers and hybridization was performed in a hybridization oven at 65°C for 17 hours with 10 rpm rotation. Finally, slides were washed using Wash Buffer Kit (Agilent Technologies) and dried at room temperature.
|
| Scan protocol |
Microarrays were scanned using the Agilent Technologies Scanner G2505C US22502723 (ChipScan software version A.8.5.1), scan region 61 x 21.6 mm, scan resolution 3 micron single pass, Green PTM 100%, no extended dynamic range (NO XDR).
|
| Description |
C2C12 cells were seeded in same numbers and cultured in same condition.
|
| Data processing |
Data was extracted from the scanned image using the Agilent Feature Extraction Software version 10.7.3.1 (protocol GE1_1200_Jun14). Quantile inter-arrays normalization was performed on the gProcessedSignal column of the result table and normalized data is reported in the VALUE column. lincRNAs (marked as “lincRNAs” in the “Description” field of platform) were not used in the normalization process and marked as “null”. Probes with Found or Well Above Background value of zero were disregarded and labeled as “null”. Mean values were calculated for multiple probes with the same Agilent Probe ID.
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| |
|
| Submission date |
Apr 28, 2017 |
| Last update date |
Dec 20, 2019 |
| 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 |
GPL10787 |
| Series (2) |
| GSE98326 |
Transcriptional changes induced by over-expression of miR-27a-3p in C2C12 myoblasts. |
| GSE98328 |
Expression Signatures of Single Isolated Myofibers of Mouse Hindlimb and miR-27a-3p or miR-142-3p overexpression in C2C12 myoblasts |
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