|
| Status |
Public on Dec 31, 2016 |
| Title |
sh69(2) |
| Sample type |
RNA |
| |
|
| Source name |
CPC-shScramble cell culture
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Sca1+ Cardiac Protenitor Cells (CPC)
genotype/variation: overexpressing sh-Scramble
|
| Treatment protocol |
Cells were transduced with respectives lentivirus at a MOI of 10 with the appropriate dilution of the stock in 3 ml of culture medium and polybrene (SIGMA) at 8 ug/ml, for seven to eight hours. Then, inoculum was removed and cell cultures were refreshed with medium before assay.
|
| Growth protocol |
Sca1+ CPC were plated in culture plates pre-coated with 0.1% gelatin and were maintained at 37°C, 3% oxygen with the corresponding medium (IMDM, 10% FCS, 1% penicillin and streptomycin and 1% L-glutamine, supplemented with 10 ng/ml EGF (E9644, Sigma), 20 ng/ml FGF (450-33, Prepotech) and LIF (103 U; ESG1107, Millipore). Medium was refreshed every 2 days. Cells were passaged at 70%-80% confluency by tripsinization.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the mirVana kit (Ambion). RNA was quantified and integrity was checked by agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
miRNA Labeling Kit (Agilent Technologies) was used to label RNA. Basically, 100 ng of total RNA were dephosphorylated and Cyanine 3-pCp molecule was ligated to the 3´ end of each RNA molecule by using T4 RNA ligase.
|
| |
|
| Hybridization protocol |
100 ng of Cy3 labelled RNA were hybridized for 20 hours at 55ºC in a hybridization oven (G2545A, Agilent) set to 15 rpm in a final concentration of 1X GE Blocking Agent and 1X Hi-RPM Hybridization Buffer, according to manufacturer's instructions (miRNA Microarray System Protocol, Agilent Technologies).
|
| Scan protocol |
Arrays were scanned at 5mm resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies) using the default settings for miRNA Microarray v2.0 (miRNA Microarray System Protocol, Agilent Technologies).
|
| Description |
CPC-shScramble, cell culture #(2)
Expression of miRNAs from Sca1+CPC overexpressing sh-Scramble
|
| Data processing |
Images provided by the scanner were analyzed using Agilent´s software Feature Extraction version 10.7.3.1 and protocol miRNA_105_Jan09
|
| |
|
| Submission date |
Mar 12, 2015 |
| Last update date |
Dec 31, 2016 |
| Contact name |
Fatima Sanchez-Cabo |
| E-mail(s) |
fscabo@cnic.es
|
| Phone |
+34 91 4531200
|
| Organization name |
CNIC
|
| Street address |
Melchor Fernandez Almagro
|
| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8824 |
| Series (1) |
| GSE66813 |
Profile of microRNAs potentially regulated by Bmi1 in Sca1+ Cardiac Protenitor Cells (CPC) |
|
