Single cell RT-PCR proceeds without the risk of genomic DNA amplification.
We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results. Thus, we demonstrated that dilutions containing a few copies of plasmid could be detected by PCR in a range which, according to the Poisson law, suggests that the PCR method can amplify from the two genomic alleles. We performed single cell RT-PCR of intronless GluR2 or GluR5 fragments by comparing cerebellar cell types where these mRNAs are known to be present or absent. For each cell the nucleus was harvested together with the cytosol. Following RT-PCR with GluR5 primers, all Purkinje cells (n = 6) yielded the expected PCR product, whereas it was not generated from any of the granule cells (n = 5). In corresponding experiments with GluR2 primers, we obtained the GluR2 product from all Purkinje cells (n = 5), but not from any of the glial cells (n = 5). These results are in agreement with the known cellular expression of GluR2 and GluR5 mRNAs. We conclude that the single cell RT-PCR method does not amplify the genomic DNA when the nucleus is aspirated together with the cytosol. We suggest that genomic DNA amplification is avoided, because the genomic alleles are not exposed during the procedure.
Johansen
F F
FF
Institut Alfred Fessard, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Lambolez
B
B
Audinat
E
E
Bochet
P
P
Rossier
J
J
D001483:Base Sequence; D002531:Cerebellum / Q000166:cytology*; D004247:DNA / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D016678:Genome; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000502:physiology; D016133:Polymerase Chain Reaction; D011689:Purkinje Cells / Q000502:physiology; D017470:Receptors, Glutamate / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D018076:DNA, Complementary; D015335:Molecular Probes; D017470:Receptors, Glutamate; D004247:DNA