siRNA: qPCR shows knockdown but Western blot shows no change (Transfection (siRNA Knockdown))

Question

Accepted Answer

qPCR confirms target mRNA is significantly reduced, but the corresponding protein band on Western blot looks unchanged. Common causes: 1) Target protein has a long half-life — protein hasn't yet declined 2) Western was performed too early after knockdown 3) siRNA only knocked down a fraction of cells 4) Western antibody sensitivity is too low to detect the residual decrease 5) Compensatory protein stabilization mechanisms Solutions: 1) Extend the post-transfection time before Western (48 – 72 h or longer) 2) Combine multiple independent siRNAs targeting the same gene 3) Confirm knockdown efficiency at the mRNA level first, then optimize WB conditions 4) If half-life is normal, audit WB conditions (antibody specificity, loading, exposure)