siRNA / miRNA gene silencing Mouse - MLO‐Y4 Lrp5

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.

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MISSION® esiRNA_esiRNA targeting mouse Lrp5 (esiRNA1)

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	To determine the involvement of specific gene products to the observed responses to sclerostin, we utilized the highly specific technique of RNA interference. We used siRNA to downregulate Car2 Lrp4 , Lrp5 , and Lrp6 expression, using a method that we described previously.30 For these experiments, MLO‐Y4 cells were seeded at a density of 2 × 104 cell per well in type I collagen–coated 24‐well plates with 0.5 mL of growth medium and incubated overnight. Cells were then transfected with either the test siRNA or nonsilencing control siRNA, using Lipofectamine 2000 transfection reagent (Invitrogen) as per the manufacturer's instructions. SiRNA sequences for mouse Car2 , Lrp4 , and the control, nonhomologous, scrambled sequence equivalent (siNEG) were purchased from Applied Biosystems (Carlsbad, CA, USA). For knockdown of mouse Lrp5 and Lrp6 , corresponding EsiRNA sequences and a nonsilencing control siRNA (siEGFP) were purchased from Sigma. Twelve hours after transfection, cells were enzymically removed from dishes using trypsin and plated onto Osteologic slides coated with calcein or uncoated, and then treated with or without rhSCL (50 ng/mL) for the assessment of pHo and Ca2+ release, as indicated. Alternatively, transfected cells were plated into wells of a 24‐well plate for gene expression analysis by RT‐PCR, as described above.

Protocol tips
To determine the involvement of specific gene products to the observed responses to sclerostin, we utilized the highly specific technique of RNA interference. We used siRNA to downregulate Car2 Lrp4 , Lrp5 , and Lrp6 expression, using a method that we described previously.30 For these experiments, MLO‐Y4 cells were seeded at a density of 2 × 104 cell per well in type I collagen–coated 24‐well plates with 0.5 mL of growth medium and incubated overnight. Cells were then transfected with either the test siRNA or nonsilencing control siRNA, using Lipofectamine 2000 transfection reagent (Invitrogen) as per the manufacturer's instructions. SiRNA sequences for mouse Car2 , Lrp4 , and the control, nonhomologous, scrambled sequence equivalent (siNEG) were purchased from Applied Biosystems (Carlsbad, CA, USA). For knockdown of mouse Lrp5 and Lrp6 , corresponding EsiRNA sequences and a nonsilencing control siRNA (siEGFP) were purchased from Sigma. Twelve hours after transfection, cells were enzymically removed from dishes using trypsin and plated onto Osteologic slides coated with calcein or uncoated, and then treated with or without rhSCL (50 ng/mL) for the assessment of pHo and Ca2+ release, as indicated. Alternatively, transfected cells were plated into wells of a 24‐well plate for gene expression analysis by RT‐PCR, as described above.

Protocol tips

To determine the involvement of specific gene products to the observed responses to sclerostin, we utilized the highly specific technique of RNA interference. We used siRNA to downregulate Car2 Lrp4 , Lrp5 , and Lrp6 expression, using a method that we described previously.30 For these experiments, MLO‐Y4 cells were seeded at a density of 2 × 104 cell per well in type I collagen–coated 24‐well plates with 0.5 mL of growth medium and incubated overnight. Cells were then transfected with either the test siRNA or nonsilencing control siRNA, using Lipofectamine 2000 transfection reagent (Invitrogen) as per the manufacturer's instructions. SiRNA sequences for mouse Car2 , Lrp4 , and the control, nonhomologous, scrambled sequence equivalent (siNEG) were purchased from Applied Biosystems (Carlsbad, CA, USA). For knockdown of mouse Lrp5 and Lrp6 , corresponding EsiRNA sequences and a nonsilencing control siRNA (siEGFP) were purchased from Sigma. Twelve hours after transfection, cells were enzymically removed from dishes using trypsin and plated onto Osteologic slides coated with calcein or uncoated, and then treated with or without rhSCL (50 ng/mL) for the assessment of pHo and Ca2+ release, as indicated. Alternatively, transfected cells were plated into wells of a 24‐well plate for gene expression analysis by RT‐PCR, as described above.

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