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Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf ,,, cMaster RTplusPCR System

Martin Simon, Petra Albrecht, Helmut J. Schmidt, and Andrs Jarrin
Martin Simon, Petra Albrecht, and Helmut J. Schmidt, University of Kaiserslautern, Germany, Department of Ecology
Andrs Jarrin, Eppendorf AG

Introduction

With the help of the Eppendorf cMaster RTplusPCR system, the surface antigen expression of Paramecium primaurelia individual cells could be shown for the first time by the detection of their mRNA. An exception to the mutual exclusion expression of these proteins could now be verified at the RNA level for the first time. This was achieved through a combination of multiplex and two-step RT-PCR.*

The cell surface of Paramecium, a free-living ciliate, is coated with high molecular weight proteins. These are known as immobilization antigens (iAg) because certain antisera against these surface proteins completely prevent the movement of the cells. The various species of Paramecium can express a series of immobilized antigens. Twelve proteins for Paramecium tetraurelia and three for Paramecium primaurelia are known. These proteins, coded by distinctive genes, are subject to "mutual exclusion expression. This means that each cell of a culture expresses the same iAg, which in turn defines the serotype of the cell. The transformation of one serotype into another is induced by changes to the ambient conditions, eg, the temperature. The coding nucleic acid sequences of the immobilization antigens protein G and D of the P. primaurelia Strain 156 are known. Two pairs of primers were designed, each being specific for one of the genes. In this way, iAg-specific transcription activity could be monitored via a
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