|
| Status |
Public on Aug 01, 2024 |
| Title |
SK-N-MC, EGFPCAG78, control-siRNA treatment (rep2) |
| Sample type |
RNA |
| |
|
| Source name |
SK-N-MC, EGFPCAG78, control-siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human neuroblastoma cell line SK-N-MC
plasmids used: EGFPCAG78
sirnas used: control-siRNA
|
| Treatment protocol |
Plasmid DNA and siRNA transfection was performed using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) and Lipofectamine RNAiMAX (13778150, Thermo Fisher Scientific), respectively.
|
| Growth protocol |
The human neuroblastoma SK-N-MC cells (HTB-10TM, American Type Culture Collection) were cultured in DMEM (11995065, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (F7524, Sigma-Aldrich) and 1% penicillin–streptomycin (15140122, Thermo Fisher Scientific).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Small RNA species from SK-N-MC cells were purified using RNeasy Mini (Qiagen) and RNeasy MinElute Cleanup (Qiagen) kits.
|
| Label |
SYBR Green
|
| Label protocol |
PCR assays were performed using the Qiagen miRCURY LNA miRNA miRNome PCR Panel following the Manufacturer’s instructions. Reverse transcription was performed using miRCURY® LNA® RT system (Qiagen). Quantitative real-time PCR were performed (QuantStudio 7, ABI) with 45 cycles at 95 °C for 15 seconds and 60 °C for 60 seconds.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
SK-N-MC cells transfected with EGFPCAG78 upon scramble control siRNA treatment
SAMPLE 11
|
| Data processing |
The Ct values were automatically generated from the ABI QuantStudio 7 Real-time PCR System.
The Ct values from target miRNAs were normalized to the Ct value of housekeeping gene U6 snRNA (ΔCt = Cttarget miRNA – CtU6 snRNA).
The ΔCt values of hsa-let-7b-5p miRNA from three sets of “Untransfected + control-siRNA” samples were averaged to obtain the ΔCtaveraged, and Ct values from each set of control and experimental group samples were further normalized to ΔCtaveraged to obtain the ΔΔCt values (ΔΔCt = Ct – ΔCtaveraged).
Target miRNA expression was calculated using 2^-ΔΔCt method.
Matrix normalized worksheet reports normalized signal (ΔΔCt values).
Fold Change worksheet reports target miRNA expression (2^-ΔΔCt method).
|
| |
|
| Submission date |
Jul 08, 2024 |
| Last update date |
Aug 01, 2024 |
| Contact name |
Ho Yin Edwin Chan |
| E-mail(s) |
hyechan@cuhk.edu.hk
|
| Phone |
+85239438032
|
| Organization name |
The Chinese University of Hong Kong
|
| Department |
School of Life Sciences
|
| Street address |
MMW509
|
| City |
Hong Kong |
| ZIP/Postal code |
000000 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL34683 |
| Series (1) |
| GSE271666 |
Real-time quantitative PCR profiling of human miRNAs in CAG RNA-expressing cells |
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