Dear All,

I am doing knockdown of the ALK in H3122 cells using Cas13. Based on the qRT-PCR result, I saw ALK is expressed significantly higher than non-targeting control. Which is the opposite of our expectation. Does anybody have this experience with siRNA or shRNA mediated ALK knockdown? or could you please tell me why this happening? what is the solution?

Background: I succeeded in the knockdown of Luciferase using Cas13. But unfortunately when I tried to knockdown of ALK in H3122 cells. It shows the opposite. For your consideration, my work was like- I did transfection with Lipofectamine 3000 and mRNA was extracted using TRIol at 48h of post-transfection. cDNA was synthesized using Oligo-dT primer from 100 ng of the RNA. Afterward, SYBR green-based qRT-PCR was done. And CT value was normalized with GAPDH.

Your valuable suggestion and cordial help are highly appreciated!!

Thanks in advance!