siRNA / miRNA gene silencing Rat - NRCM Klf15

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. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

1 Matching solution
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

ON-TARGETplus Rat Klf15 (85497) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

Protocol tips
1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

Protocol tips

1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment