SINGLE-CELL RT-PCR PROCEEDS WITHOUT THE RISK OF GENOMIC DNA AMPLIFICATION

Citation
Ff. Johansen et al., SINGLE-CELL RT-PCR PROCEEDS WITHOUT THE RISK OF GENOMIC DNA AMPLIFICATION, Neurochemistry international, 26(3), 1995, pp. 239-243
Citations number
11
Categorie Soggetti
Biology,Neurosciences
ISSN journal
01970186
Volume
26
Issue
3
Year of publication
1995
Pages
239 - 243
Database
ISI
SICI code
0197-0186(1995)26:3<239:SRPWTR>2.0.ZU;2-I
Abstract
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.