siRNA / RNAi /miRNA transfection Human Cells - HT-1376 CD74

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid-mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time-consuming, but provide a more permanent expression of RNAi in the cells and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines.

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4 years ago

4 years ago by Keith L. Morrison Canada

siRNA/RNAi/miRNA transfection human

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

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Found 1 matching solution for this experiment

CD74 siRNA and shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection.	The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h.	Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells.

Upstream tips
Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection.
Protocol tips
The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h.
Downstream tips
Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells.

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