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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 RT-PCR strategy.

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.

Methods

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.

RT-PCR
For both one- and two-step RT-PCR, the reactions were prepared directly in the tubes in which the poly-A-RNA had been fixed. The RNA template is therefore missing in the protocols.

Three different primer pairs were used:

Serotype G: Product app. 800 bp
Serotype D: Product app. 500 bp
Control primer of non-transcribed genomic region: Product app. 400 bp
Amplification reactions were run as duplicates.

One-step RT-PCR

Preparation

Components Volume Final concentration in the reaction dNTP mix (each 10 mM) 1.0 l 200 M Forward primer G (100 M) 1.0 l 2 M Reverse primer D (100 M) 1.0 l 2 M RTplusPCR buffer with 25 mM Mg++ 5.0 l 1 x RNase inhibitor (0.5 g/l) 0.5 l 5 ng/l cMaster RT enzyme (15 U/l) 0.5 l 0.15 U/l cMaster PCR enzyme mix (5 U/l) 0.5 l 0.05 U/l RNase-free water 40.5 l Total volume 50.0 l


Program parameters of the one-step RT-PCR

Step Temp. Duration cDNA synthesis 50C 50 min Initial denaturation 94C 2 min Denaturation 94C 20 s Annealing 45C 20 s 40x Elongation 68C 30 s Cooling 4C Hold
Two-step RT-PCR

Gene-specific primers were used for the cDNA synthesis.

Reverse transcription preparation

Components
Volume Final concentration in the reaction dNTP mix (each 10 mM) 2.0 l 1 mM Reverse primer G (100 M) 1.0 l 5 M Reverse primer D (100 M) 1.0 l 5 M Left control primer (100 M) 1.0 l 5 M RNase-free water 5.0 l Master mix 1 10.0 l RTplusPCR buffer with 25 mM Mg++ 4.0 l 2 x RNase inhibitor (0.5 g/l) 0.5 l 12.5 ng/l cMaster RT enzyme (15 U/l) 2.0 l 1.5 U/l RNase-free water 3.5 l Master mix 2 10.0 l
Program parameters of the reverse transcription

Step Temp. Duration Template RNA denaturation 65C 5 min Fixation of the RNA 0C 5 min Addition of master mix 2 (10 l) Primer annealing 45C 10 min cDNA synthesis 50C 50 min
Preparation of the PCR


Components Volume Final concentration in the reaction dNTP mix (each 10 mM) 2.0 l 400 M Forward primer D (100 M) 0.5 l 1 M Reverse primer D (100 M) 0.5 l 1 M Forward primer G (100 M) 0.5 l 1 M Reverse primer G (100 M) 0.5 l 1 M Right control primer (100 M) 0.5 l 1 M Left control primer (100 M) 0.5 l 1 M First-strand cDNA, see above 6.0 l Master mix 3 11.0 l RTplusPCR buffer with 25 mM Mg++ 5.0 l 1 x cMaster PCR enzyme mix (5 U/l) 0.5 l 0.05 U/l RNase-free water 33.5 l Master mix 4 39.0 l 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 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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