When using the Eppendorf cMaster RTplusPCR system, this method could be adapted to individual cells of P. primaurelia for the first time. Other RT-PCR systems, which use AMV reverse transcriptase, enable comparable results only with the cells of P. tetraurelia (data not shown). P. tetraurelia cells are considerably larger and contain significantly more RNA. The cMaster RTplusPCR system, which uses a genetically and posttranslationally modified reverse transcriptase, is characterized by its much greater sensitivity.
All individual steps, consisting of mRNA isolation, reverse transcription and PCR, were carried out on the same day, making it unnecessary to freeze or store intermediate products for longer periods of time. All glassware was RNase deactivated. Micro test tubes, pipette tips and solutions were used in RNase-free quality.
Isolation of the mRNA from single cells of P. primaurelia
A "capture" strategy based on biotin/streptavidin was chosen for the isolation of mRNA from single cells. Single cells were lysed in 20 l buffer by pipetting quickly up and down. Following the addition of a biotin-labeled oligo dT-probe to the lysate, the mixture was incubated at 37C for 10 minutes (hybridization of probe and mRNA).
The solution was then transferred into streptavidin-coated micro test tubes, and the incubation was repeated. As a result, the biotin-labeled mRNA/probe hybrids bind tightly to the micro test tube, minimizing losses. The tube was then rinsed three times with a wash buffer in order to minimize DNA contamination.
For both one- and two-step RT-PCR, the reactions were prepared directly in