We have previously described a method for detection of mRNAs expressed
in single cells after patch-clamp recordings. The method, termed sing
le cell RT-PCR, involves aspiration of the cell content, a reverse tra
nscription (RT) step, and a polymerase chain reaction (PCR) using spec
ific 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, sug
gests that the PCR method can amplify from the two genomic alleles. We
performed single cell RT-PCR of intronless GluR2 or GluR5 fragments b
y comparing cerebellar cell types where these mRNAs are known to be pr
esent 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 w
ith GluR2 primers, we obtained the GluR2 product from all Purkinje cel
ls (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 GluR
5 mRNAs. We conclude that the single cell RT-PCR method does not ampli
fy the genomic DNA when the nucleus is aspirated together with the cyt
osol. We suggest that genomic DNA amplification is avoided, because th
e genomic alleles are not exposed during the procedure.