I silenced a gene by RNAi method, for that i chose the gene and the size of the gene is 1.5kb and my insert size is 500nts. i did the transfection and i want to confirm the silencing by northern analysis. when i did i got the ribosomal RNA instead of my specific mRNA reduction and dsRNA accumulation.
Total RNA was prepared with TRIzol reagent (Sigma), and 20 μg/lane was fractionated on a 1.2% agarose, 2.2 M formaldehyde gel. The RNA was visualized with ethidium bromide. The RNA was transferred to a nylon membrane (Hybond; Amersham Biosciences) for 14 hrs and probed with [α-32P] UTP-labeled used SSC for transfer and Prehybridization at 55 for 2hrs incubation, in the probe for 10hrs and Washing at 60 for 2X SSC 20 mins two times.
In this image you can the samples TET induced and uninduced RNA. but the result was confusing and am getting such a strong ribosomal RNA (2.2 Kb) contamination.