Abstract
To detect the Ki-ras oncogene expression in paraffin embedded sections by the in situ RT-PCR method, experimental procedure, including fixation of tissue, protease or DNase digestion and cycling conditions, was examined in detail with paraffin embedded mouse liver sections using a set of DNA primers for Ki-ras oncogene m-RNA and rTth DNA polymerase. A mouse was perfused via the left ventricle with 20ml phosphate buffered saline (PBS) followed with 50ml of 10% formalin in PBS under deep anesthesia. The liver was dissected into 5×5×2mm block and further fixed in the same fixative at 4℃ for 16 hrs. The block was then embedded in paraffin and sectioned at a thickness of 5μm. The section was digested with pepsin (2mg/ml, in 0.1N HCl) at 37℃ for 6 to 7min, then treated with DNase (1 unit/μl) at room temperature for 18 hours, and fixed again in the same fixative. The section was then processed by the in situ RT-PCR procedure using Ki-ras primers, rTth polymerase and digoxigenin-11-dUTP. The resulting specimen was heated at 94℃ for 3min and 57℃ for 60min for RT followed by 20 cycles of PCR reaction ; 94℃ for 1min, 55℃ for 1min and 65℃ for 40sec. The section was then treated with anti-DIG-antibody HRP conjugates and stained with DAB. This procedure provides reprodusible result with high contrast to the background.
