SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.
Total time
~3-4 days (transfection + protein extraction + analysis)
Steps
1
Design binding domains for target mRNAs
Construct SINEUP antisense RNA with binding domain targeting specific mRNA sequences. The binding domain determines target specificity for translation enhancement.
▶ 00:48
2
Culture cells and transfect SINEUP constructs
Grow cultured cell lines and introduce SINEUP RNA via transfection. Allow sufficient incubation time for RNA uptake and target interaction.
▶ 01:48
3
Extract total protein from transfected cells
Lyse transfected cells and isolate total protein lysate for downstream analysis. Quantify protein concentration before separation.
▶ 03:46
4
Separate proteins and detect by Western blot
Run SDS-PAGE to separate proteins by molecular weight, transfer to membrane, and detect target proteins using antibodies. Quantify band intensity.
▶ 05:32
5
Quantify translation upregulation by imaging
Use semi-automated high-throughput imaging system to detect SINEUP-tagged proteins in fixed cells. Measure fluorescence intensity and protein expression levels.
▶ 07:21
6
Analyze translation upregulation results
Compare target protein levels between SINEUP-treated and control samples. Calculate fold-change in translation efficiency.
▶ 08:26