|
| Status |
Public on Feb 20, 2017 |
| Title |
NRVM KChIP2 siRNA treated |
| Sample type |
RNA |
| |
|
| Source name |
isolated ventricular myocytes from neonatal rats (1-2 days old) that were treated with KChIP2 siRNA
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: isolated cardiac ventricular myocytes
strain: Sprague Dawley
age: neonatal (1-2 days old)
|
| Treatment protocol |
Knockdown of KChIP2 was conducted by transfecting with siRNA for KChIP2 (Ambion, Cat#: 4390771, ID: s132782), or a scrambled siRNA control (Ambion, Cat#: 4390843). 180 pmol of siRNA was transfected using 15 μL of Lipofectamine 2000 reagent according to the manufacturer’s instructions. Following the transfection period, media was changed to DMEM/5% FBS/penicillin/streptomycin. Cells were cultured for 72 hrs total before collection for total RNA, with a media change once after 48 hrs of culture.
|
| Growth protocol |
Rat neonatal ventricular myocytes were isolated 1-2 days after birth. Briefly, hearts were minced in HBSS, and tissue fragments were digested overnight with trypsin at 4 °C. Trypsinized fragments were treated repeatedly for short periods of time with collagenase at 37 °C followed by trituration. Dissociated cells were pre-plated for 2 hrs at 37 °C in DMEM supplemented with 5% fetal bovine serum (FBS) and penicillin/streptomycin. NRVMs were collected and replated with 1.5 x 10^6 cells per 35 mm dish in DMEM/5% FBS/penicillin/streptomycin with 0.1 mM bromodeoxyuridine (BrdU) to suppress fibroblast growth and maintained at 37 °C, 5% CO2. These conditions were maintained for 24-36 hrs, after which control or KChIP2 siRNA was delivered. Cells were left an additional 72 hrs before collection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated RNA were prepared according to Affymetrix FlashTag Biotin HSR RNA Labeling Kit from 1 ug total RNA
|
| |
|
| Hybridization protocol |
The labeled RNA were hybridized for 16 hr at 48C on GeneChip miRNA Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
miRNA expression data (log2 scale) from isolated neonatal rat ventricular myocytes
|
| Data processing |
The data were analyzed with ExpressionConsole version 1.4.1.46 using the Affymetrix MicroRNA Arrays - RMA+DABG-Rat_only analysis with default settings and global scaling as normalization method to identify the log2 probe intensity values.
|
| |
|
| Submission date |
Dec 08, 2015 |
| Last update date |
Feb 20, 2017 |
| Contact name |
Isabelle Deschenes |
| E-mail(s) |
isabelle.deschenes@case.edu
|
| Organization name |
Case Western Reserve University
|
| Street address |
2500 Metrohealth Drive
|
| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44109 |
| Country |
USA |
| |
|
| Platform ID |
GPL19117 |
| Series (1) |
| GSE75806 |
miRNA expression data from neonatal rat ventricular myocytes (NRVM) silenced for KChIP2 |
|
