In loss-of-function experiments, we knocked down Pkm, Pkm1 and Pkm2 in C2C12 myotubes using silencer RNA (Ambion, Carlsbad, CA, USA, see Table 2 for siRNA sequences). C2C12 myoblasts were grown and differentiated as described. On day 6, myotubes were transfected with siRNA targeted against Pkm, Pkm1 or Pkm2 using the liposome-mediated method (lipofectamine RNAiMAX, Invitrogen, Cat# 13778100, Carlsbad, CA, USA). As a negative control, a non-targeting silence RNA sequence (siControl) was used. siRNA was diluted in Opti-MEM medium and incubated for 5–10 min with lipofectamine mixture. RNA–lipofectamine complexes with a final concentration of 20 nM were added to each well. On day 7, the differentiated myotubes were treated with IGF-1 (100 ng/mL, Peprotech, Cat#100-11, London, UK) and harvested at day 8 (48 h post-transfection).
Silencer rna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Silencer RNA is a laboratory product designed for RNA interference (RNAi) experiments. It is used to silence or reduce the expression of specific genes in cells. Silencer RNA functions by targeting and degrading the complementary messenger RNA (mRNA) molecules, thereby preventing the translation of the target gene into protein.
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Lab products found in correlation
2 protocols using silencer rna
1
Knockdown of Pkm, Pkm1, and Pkm2 in C2C12 Myotubes
2
PHGDH Knockdown in Myotubes
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To carry out a PHGDH loss-of-function experiment, we knocked down Phgdh in C2C12 and primary myotubes using silencer RNA (Ambion, Carlsbad, CA, USA, see Table 2 for siRNA sequences). C2C12 myoblasts or primary myoblasts were grown and differentiated as described. On day 6, myotubes were transfected with siRNA targeted against Phgdh using the liposome-mediated method (Lipofectamine RNAiMAX, Invitrogen, Cat# 13778100, Carlsbad, CA, USA). As a negative control, a non-targeting silence RNA sequence (siControl) was used. siRNA was diluted in Opti-MEM medium and incubated for 5-10 minutes with Lipofectamine mixture.
RNA-lipofectamine complexes with a final concentration of 20 nM were added to each well. On day 7, the differentiated myotubes were treated with IGF-1 (100 ng ml-1) and harvested at day 8 (48 hours post-transfection).
RNA-lipofectamine complexes with a final concentration of 20 nM were added to each well. On day 7, the differentiated myotubes were treated with IGF-1 (100 ng ml-1) and harvested at day 8 (48 hours post-transfection).
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