I am trying to knockdown basal level expression of my target microRNA by transfected Anti-Mir,miRNA Inhibitor, but I didn’t get significant  level of knockdown  result by real time PCR.

Have any one any idea please help me?

Conditions.

·         Cell line-MCF12A (The MCF-12A cell line is a non-tumorigenic epithelial cell line). For cell culture I am using  MEGM, Kit Catalog No. CC-3150 by  Lonza/Clonetics with 100 ng/ml cholera toxin.

·         Name of the transfection reagent- lifofectamin 2000(forward transfection),  RNAiMAX(reverse transfection). Transfection down by according to the thermos fisher scientific. I did also forward and reverse but results is same after 24h and also 48h of incubation.

·         For the RNA isolation, used miRCURY RNA isolation kit(EXICON). For cDNA making, used miRCURY LNA universal RT microRNA PCR/Universal cDNA Synthesis kit(exiqon), for real Time PCR, used miRCURY LNA universal RT microRNA PCR/ExiLENT SYBER Green(exiqon). For endogenous control I used U6. Everything I did according to company protocol.