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

for coexpression of both genes in the individual cells were carried out using the two-step multiplex RT-PCR strategy (Fig. 2). In addition to the G and D protein genes, the fragment of a non-transcribed genomic region was also coamplified.

In the two-step multiplex RT-PCR format, it was possible to detect the presence of the mRNAs of the proteins D and G in individual cells of P. primaurelia. The mRNA specificity of the RT-PCR products could be proven by the absence of the genomic control amplicon (see Fig. 2, lanes 1+2 and 3+4). These findings represent an exception to the previously assumed exclusive expression of both genes in individual cells. This analysis carried out at the RNA level could be confirmed by immune fluorescence staining at the protein level.

These results show that molecular analyses of smallest sample quantities require the use of suitable reagents compellingly. Only the superior sensitivity of the Eppendorf cMaster RTplusPCR system made possible the establishment of this RT-PCR assay from single cells of P. primaurelia.

* This product is sold under licensing arrangements with F. Hoffman-La Roche Ltd., Roche Molecular Systems, Inc. and Applied Biosystems.





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