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

Abstract

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 ¼ 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.

Laser capture microdissection and single-cell RT-PCR without RNA purification

“…Extension of LCM from small populations of cells to individual cells has relied on PCR amplification of cell DNA (Suarez-Quian et al, 1999;Obiakor et al, 2002;Orba et al, 2003) or the perceived need to purify the very small amount of RNA, approximately 20 pg from a single captured cell for RT and PCR (Jin et al, 2001;Parlato et al, 2002;Michel et al, 2003;Kamme et al, 2004;Lu et al, 2004;Fassunke et al, 2004). Hydraulic microdissection has also been coupled with nested-PCR amplification of genomic DNA to identify sequences in single B cells (Obiakor et al, 2002).…”

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

“…However, as formalin fixation results in fragmentation of RNA, these archives are difficult to exploit using recently developed molecular tools that allow a comprehensive analysis of gene expression patterns. [1][2][3] This has increased the efforts in recent years to establish biobanks of nonfixed frozen tissue. Together with attached relevant clinical information, these biobanks can provide researchers a reliable, organised source of human tissues for RNA-based analysis.…”

Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment

“…This technique has been devised to isolate distinct cell populations from their natural environment in situ not only according to morphology but also to immunophenotype criteria. [21][22][23] It has been used successfully for PCR-based gene analysis of specific cell populations in brain tissue, [24][25][26] brain vessels 27 and epithelial cells, 28,29 but to our knowledge has never been employed for genome-wide discovery. A major limitation has been the necessity to procure high quality and sufficient quantity of RNA.…”