( Program paramete...)Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf ,,, cMaster RTplusPCR System

Program parameter of the PCR

Step
Temp. Duration Initial denaturation 94C 2 min Denaturation 94C 20 s Annealing 45C 20 s 35x Elongation 68C 30 s Cooling 4C Hold

Results

Initially, the RT-PCR from individual cells of P. primaurelia were established in the one-step procedure. Only one primer combination was used for serotype G or D.

Fig. 1: One-step RT-PCR from individual cells of P. primaurelia. G and D label the RT-PCR products of the serotypes G and D. M: 100 bp ladder. Fig. 2: Two-step multiplex RT-PCR from single cells of P. primaurelia. 10 ng of genomic DNA were added to each of the samples from lanes 1+2 during the RT step. G and D mark the RT-PCR products of the serotypes G and D, and * marks the genomic control amplicon. M: 100 bp ladder.

When using the Eppendorf cMaster RTplusPCR system, the transcripts of the serotypes D and G were clearly detectable using the one-step RT-PCR procedure (Fig. 1). The intensity differences of the amplified DNA can probably be attributed to individual differences in expression of the genes in individual cells.

The experiments
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