In situ-RT and immunolaser microdissection for mRNA analysis of individual cells isolated from epilepsy-associated glioneuronal tumors

Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy ( n = 6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription ( in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short-vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance ( glyceraldehyde dehydrogenase; GAPDH), medium-abundance ( glial fibrillary acidic protein; GFAP), or low abundance ( neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types ( myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.