MISSION® esiRNA_esiRNA targeting mouse Lrp6 (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.

Publication protocol

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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