Expression level changes of imprinted genes in macroH2A1 knockdown cell lines. A) The expression levels were measured by RT-PCR with fixed number of cycles ranging from 30 to 38. The cycle numbers for Zim1 and Tsix were 38 and 36, respectively, and the cycle numbers for all the remaining genes were 34. The amplified products from four different cell lines were separated on agarose gels: lane 1, the Neuro2A cells with no transfection (NT); lane 2, the transfectants with the empty pSicoR vector (pSicoR); lane 3, the stable transfectant #2; and lane 4, the stable transfectant #11. Individual genes are grouped together based on their chromosomal locations (Peg3 and Xist/Tsix domains) and their purposes (Side Effects and Control). GAPDH, β-actin and 28S were used as quantitative controls. Four genes, including p53, IFITM1, Oas2 and Mx1, were used for monitoring potential toxic and side effects caused by siRNA experiments. B) Quantitative real time PCR analysis of the genes located within the Peg3 domain. The genomic structure of the Peg3 domain illustrated with gene names, lines and arrows. Gene names in red color represent paternally expressed genes while those in blue color represent maternally expressed genes. The expression levels of each gene were first normalized with two different control genes, GAPDH and 28S, and later compared with the normalized level of the cells without transfection (NT). The values in graphs are the averaged fold differences relative to those of the non-transfected cells (NT) with standard deviations (S.D.). We performed this experiment at least three times from RNA isolation to real time qRT-PCR.