Single-cell mRNA Research Articles

Abstract A63: Identification and molecular characterization of single disseminated metastasis progenitor cells in prostate cancer: The significance of EpCAM-positive cells

Abstract Carcinoma of the prostate is the most frequently diagnosed cancer and the second leading cause of cancer deaths in males. While it can be detected early, its progression is variable and patients that will benefit from aggressive therapy are hard to identify, leading to considerable overtreatment. Nevertheless, a fraction of patients will develop metastasis years after surgery and finally succumb to the disease. The detection of early disseminated cancer cells (DCCs) in bone marrow (BM) of carcinoma patients without distant metastasis is associated with poor prognosis. DCCs comprise precursor cells of later arising metastasis, and they are direct targets for adjuvant therapies, aiming to delay or prevent outgrowth of macrometastasis. DCCs are extremely rare and can be detected in BM by using antibodies against epithelial markers, such as cytokeratins (CKs) and epithelial cell adhesion molecule (EpCAM). We have previously observed that CK+ DCCs in BM detected in samples taken before surgery are associated with poor survival of prostate cancer patients. In contrast, we found that the detection of persisting CK+ cells in BM samples up to 8 years after surgery had no impact on the survival of prostate cancer patients, indicating that prostate cancer can become a chronic, non-progressive disease. This result prompted us to search for cells that persist after surgery and are associated with metastatic progression. We therefore tested the EpCAM in a cohort of 220 prostate cancer patients. We directly compared the prognostic impact of CK+ and EpCAM+ DCCs detected in BM samples taken months to years after surgery. Strikingly, while CK+ cells were, as previously shown, not informative about progression, the detection of EpCAM+ DCCs after surgery was associated with poor survival. Furthermore, double staining against both epithelial markers identified different subsets of DCCs. The analysis of single DCCs from BM by single cell comparative genomic hybridization (SCOMP) demonstrated that both CK+ and EpCAM+ cells are tumor cells. Interestingly, EpCAM+ cells harbored significantly more genomic aberrations than CK+ cells and are defined by characteristic changes. Since staining for EpCAM enables isolation of single cell's mRNA, we have isolated EpCAM+ cells from an additional cohort of 108 patients and from 8 healthy donors. Total genomic DNA and mRNA from these single cells were subjected to combined analysis of genome and transcriptome. PCR analysis of epithalial markers confirmed the existence of different subpopulations of EpCAM+ cells. Furthermore, using microarray analysis, we have obtained gene expression signatures of different subpopulations of DCCs. Finally, using prostate epithelial cell lines, we have investigated the functional differences of different subpopulations of EpCAM+ cells. Our results indicate an unexpected diversity of early-disseminated cancer cells displaying different phenotypes and genotypes and divergent metastatic potential. Gene expression analysis revealed great heterogeneity of the expression of analyzed markers within cells isolated from patients' BM samples. This suggests that different subpopulations of EpCAM+ DCCs may exist in diverse functional states, and may have different functional relevance. Citation Format: Miodrag Gužvić, Bernhard Polzer, Roman Ganzer, Dorothea Weckermann, Bernhard Braun, Inka Appel, Matthias Maneck, Wolfgang Wieland, Christoph A. Klein. Identification and molecular characterization of single disseminated metastasis progenitor cells in prostate cancer: The significance of EpCAM-positive cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A63.

Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

We investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen, Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by the EPA gene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ∼95% of adherence to epithelial cells in vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression in C. glabrata BG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference in EPA1 sequence or (sub)telomere length and was overcome by ectopic SIR3 expression. Moreover, differences between strains in the silencing dependence of EPA1 expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains where EPA1 was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties of C. glabrata cell populations, with potential implications for microbial pathogenesis more broadly.

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I ATP) in guinea pig myenteric neurons. I ATP was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01–10 mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01–0.3 mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC 50 values of these phases were 38 and 1759 μM, respectively. 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01–1 mM), and less potent (EC 50 142 μM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1 mM) and for BzATP-induced currents (0.1 mM; I BzATP) but a significant lower rectification was noticed when lsP2X receptors were activated (5 mM). Brilliant blue G (BBG) at a concentration of 0.3 μM (known to inhibit only P2X7 receptors) reduced I ATP when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I BzATP. However, hsP2X receptors and I BzATP were both inhibited by concentrations ≥ 1 μM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.

Role of Cell-to-Cell Variability in Activating a Positive Feedback Antiviral Response in Human Dendritic Cells

In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI), which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop.

Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells

Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.

Nuclear Origins of Cell-to-Cell Variability

During the past decade, it has become increasingly evident that there is variation in the transcriptome of genetically identical cells, even when grown in homogenous environments. This cell-to-cell variability has been shown to have a central role in processes ranging from stem cell differentiation to chemotherapy resistance. Given that many genes display extensive heterogeneity in their messenger RNA (mRNA) abundance on a per cell basis, understanding the nuclear sources of this variability is important for our fundamental grasp of nuclear function and stands to have clinical manifestations. In this chapter, we assess the contribution of different transcription regimes, nuclear architecture dynamics, RNA polymerase elongation, and gene copy number to transcriptome heterogeneity. We also discuss techniques that can be used to quantify single-cell mRNA abundance and conclude by commenting on future research directions.

Efficacy, Safety, and Tolerability of Low-Dose Hormone Therapy in Managing Menopausal Symptoms

Use of the lowest clinically effective dose of postmenopausal hormone therapy conforms to current recommendations and good clinical practice. Although accumulating evidence demonstrates the efficacy and tolerability of low hormone therapy doses, data about their use are limited by a lack of long-term, randomized studies. This review evaluates current evidence on the efficacy, safety, and tolerability of these preparations and their role in menopausal management.

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumour-infiltrating lymphocytes obtained from in vivo liver tumours.

We performed a genomic study combining single-cell mRNA differential display and RNA subtractive hybridization to elucidate CD8 T-cell quiescence/ignorance. By comparing actively maintained quiescent CD8 T cells from liver tumour tumour-infiltrating lymphocytes (TILs) with quiescent T cells at the single-cell level, we identified differentially expressed candidate genes by high-throughput screening and comparative analysis of expressed sequence tags (ESTs). While genes for the T-cell receptor, tumour necrosis factor (TNF) receptor, TNF-related apoptosis inducing ligand (TRAIL) and perforin were down-regulated, key genes such as Tob, transforming growth factor (TGF)-beta, lung Krüpple-like factor (LKLF), Sno-A, Ski, Myc, Ets-2 repressor factor (ERF) and RE1-silencing transcription factor (REST/NRSF) complex were highly expressed in the quiescent TIL CD8 cells. Real-time polymerase chain reaction (PCR) further confirmed these results. A regulation model is proposed for actively maintained quiescence in CD8 T cells, including three components: up-regulation of the TGF-beta pathway, a shift in the MYC web and inhibition of the cell cycle.

Imaging Target mRNA and siRNA‐Mediated Gene Silencing In Vivo with Ribozyme‐Based Reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

Expression of islet-specific microRNAs during human pancreatic development

During pancreatic islet development, sequential changes in gene expression are known to be necessary for efficient differentiation and function of the endocrine pancreas. Several studies till now have demonstrated that microRNAs (miRNAs), which regulate translation of gene transcripts, influence gene expression cascades involved in pancreas development. Some of these miRNAs; miR-7 and miR-375 have been known to be expressed at high levels in pancreas and are also known to be involved in Zebrafish pancreas development as well as insulin secretion in mice. We demonstrate here that 4 different islet-specific microRNAs (miR-7, miR-9, miR-375 and miR-376) are expressed at high levels during human pancreatic islet development. Of these, miR-375, is seen to be differentially expressed in human islet β- as well as non-β-cells. Though no significant difference in abundance of miR-375 was noted in either cell type, analysis of islet-specific miRNA and mRNA in single cells show that non-β cells contain higher levels of miR-375. Our data demonstrate that miRNAs that are known to be regulated during Zebrafish pancreatic development may play similar role in human pancreatic islet development.

Modelling and measuring single cell RNA expression levels find considerable transcriptional differences among phenotypically identical cells.

BackgroundPhenotypically identical cells demonstrate predictable, robust behaviours. However, there is uncertainty as to whether phenotypically identical cells are equally similar at the underlying transcriptional level or if cellular systems are inherently noisy. To answer this question, it is essential to distinguish between technical noise and true variation in transcript levels. A critical issue is the contribution of sampling effects, introduced by the requirement to globally amplify the single cell mRNA population, to observed measurements of relative transcript abundance.ResultsWe used single cell microarray data to develop simple mathematical models, ran Monte Carlo simulations of the impact of technical and sampling effects on single cell expression data, and compared these with experimental microarray data generated from single embryonic neural stem cells in vivo. We show that the actual distribution of measured gene expression ratios for pairs of neural stem cells is much broader than that predicted from our sampling effect model.ConclusionOur results confirm that significant differences in gene expression levels exist between phenotypically identical cells in vivo, and that these differences exceed any noise contribution from global mRNA amplification.

Static Compression of Single Chondrocytes Catabolically Modifies Single-Cell Gene Expression

Previous work has established that mechanical forces can lead to quantifiable alterations in cell function. However, how forces change gene expression in a single cell and the mechanisms of force transmission to the nucleus are poorly understood. Here we demonstrate that the gene expression of proteins related to the extracellular matrix in single articular chondrocytes is modified by compressive forces in a dosage-dependent manner. Increasing force exposure catabolically shifts single-cell mRNA levels of aggrecan, collagen IIa, and tissue inhibitor of metalloproteinase-1. Cytohistochemistry reveals that the majority of strain experienced by the cell is also experienced by the nucleus, resulting in considerable changes in nuclear volume and structure. Transforming growth factor- β1 and insulin-like growth factor-I offer mechanoprotection and recovery of gene expression of aggrecan and metalloproteinase-1. These results suggest that forces directly influence gene transcription and may do so by changing chromatin conformation.

Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

BackgroundGene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells.ResultsLow concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined.ConclusionNoise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.

Cajal–Retzius cells switch from expressing γ‐less to γ‐containing GABAA receptors during corticogenesis

Cajal-Retzius cells are implicated in regulating neuronal migration and lamination during corticogenesis. In rodents, Cajal-Retzius cells are transient, being prevalent in the marginal zone of the embryonic neocortex and declining over the first two postnatal weeks. While studies have examined in postnatal neocortex the properties of GABA(A) receptors in Cajal-Retzius cells, less is known about their disposition at embryonic stages. Here, we combined patch-clamp electrophysiology and single-cell mRNA profiling to probe the expression of GABA(A) receptors in Cajal-Retzius cells. In embryonic neocortical slices, GABA elicited GABA(A) receptor-mediated current responses that were diazepam-insensitive and inhibited by Zn(2+), a pharmacological profile consistent with expression of gamma-less GABA(A) receptor isoforms. Non-Cajal-Retzius cells in the same embryonic slices, on the other hand, were robustly potentiated by diazepam and were insensitive to Zn(2+), typical of gamma-containing GABA(A) receptor isoforms, as were Cajal-Retzius cells in the postnatal neocortex. Single-cell mRNA profiling and immunohistochemistry confirmed expression of GABA(A) receptor gamma subunit transcript and protein, respectively, in individual reelin-expressing cells in the postnatal cortex but not in their embryonic counterparts. We conclude that Cajal-Retzius cells express gamma-less GABA(A) receptors at embryonic stages and switch to expressing gamma-containing GABA(A) receptor isoforms during postnatal neocortical development.

Chance Fluctuations in mRNA Output in Mammalian Cells

In the drama of cell biology, both genetics and environment write the script, and chance throws in the twists of plot. In general, most cells live relatively predictable lives: divide, differentiate, and die. Yet chance leaves its imprint even in ordinary cells. For instance, bacterial or yeast cells in culture are known to produce widely different amounts of certain proteins, even when they are genetically identical. Scientists attribute such cell-to-cell variations to chance fluctuations in the cells’ ability to make these proteins. They also speculate that such fluctuations may benefit the cells in their struggle to adapt and survive. But opinions vary as to which step in protein production is subject to random fluctuations. Proteins result from the translation of messenger RNAs (mRNAs), which come from the transcription of genes. Fluctuations in protein output could reflect a whimsy intrinsic to the expression of the coding gene, or random swings in global, or extrinsic, regulators of the gene’s expression. In yeast, extrinsic causes, such as variations in cellular volume, seem to predominate. But in a new study, Arjun Raj and his colleagues show that in mammalian cells, intrinsic causes, specifically the genes’ ability to transition randomly between active and inactive transcriptional states, can be crucial. The researchers developed a sensitive technology to detect mRNAs in single cells and examined the output of a genetically engineered reporter gene they called M1, which they had introduced into Chinese hamster ovary (CHO) cells. After staining cultures of identical cells with a fluorescent probe specific to M1, they counted fluorescent dots corresponding to single M1 mRNA molecules in individual cells. At any given time, a few cells displayed a large, bright cluster of these spots, indicative of recently transcribed mRNAs still densely packed around the M1 gene. These cells also had the largest number of M1 mRNAs (over 150). In these cells, the M1 gene was therefore actively churning out mRNAs. But the majority of cells displayed fewer than 50 M1 mRNAs that were dispersed over their entire volume. In these cells, the M1 gene had become silent. These results showed that the M1 gene is expressed via infrequent bursts of transcriptional activity in the CHO cells. If these bursts reflected the uneven distribution of transcription factors among CHO cells, then multiple reporter genes in a single cell should burst at the same time. The researchers generated cell lines that contained two versions of the reporter gene (M1 and M2) that were controlled by the same transcriptional activators but whose mRNAs could be distinguished with probes of different colors. In some lines, the two genes had landed in separate genomic locations. The researchers found that they burst independently of each other. They concluded that bursts are not induced by extrinsic factors such as M1- or M2-specific transcriptional activators. Rather, they reflect the intrinsically random ability of both the M1 and M2 genes to switch between an inactive and an active state. Nevertheless, when the two versions of the reporter gene landed next to each other, they did burst in synchrony, as if switching spread locally among contiguous genes. This observation suggests that switching follows the waves of condensation and decondensation that randomly breathe through the coils of genomic DNA (chromatin). Indeed, genes are mostly silent when packed into dense chromatin. But when they decondense, the transcription machinery can latch on to their DNA and begin making RNA. Raj and his colleagues propose that genes switch to an active state as a consequence of randomly initiated decondensation, and that transcription factors merely stabilize their active state. Consistent with this proposal, they find that reducing the availability of a transcriptional factor, or increasing M1’s affinity for this factor, does not significantly increase the frequency of bursts. Only the size of the bursts—that is, the amount of RNA produced in each burst—increases. Random bursting is not restricted to artificial genes such as M1. The researchers document the same behavior in the gene encoding RNA polymerase, the lead actor of transcription. That cells would tolerate chance fluctuations in an mRNA so fundamental to their survival comes as a surprise. The researchers show that in the case of the M1 gene, protein stability buffers the consequences of erratic mRNA production. This is because stable proteins are only “topped up” by the occasional bursts of transcription, whereas unstable proteins follow the variations in mRNA levels more closely. This finding indicates that protein stability may be a critical factor in the cell’s ability to tolerate variations in transcription. Whether chance fluctuations in RNA production sometimes produce beneficial twists of fate for mammalian cells remains to be shown.

Microfluidic Single-Cell mRNA Isolation and Analysis

Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.

Real-Time Kinetics of Gene Activity in Individual Bacteria

Protein levels have been shown to vary substantially between individual cells in clonal populations. In prokaryotes, the contribution to such fluctuations from the inherent randomness of gene expression has largely been attributed to having just a few transcripts of the corresponding mRNAs. By contrast, eukaryotic studies tend to emphasize chromatin remodeling and burst-like transcription. Here, we study single-cell transcription in Escherichia coli by measuring mRNA levels in individual living cells. The results directly demonstrate transcriptional bursting, similar to that indirectly inferred for eukaryotes. We also measure mRNA partitioning at cell division and correlate mRNA and protein levels in single cells. Partitioning is approximately binomial, and mRNA-protein correlations are weaker earlier in the cell cycle, where cell division has recently randomized the relative concentrations. Our methods further extend protein-based approaches by counting the integer-valued number of transcript with single-molecule resolution. This greatly facilitates kinetic interpretations in terms of the integer-valued random processes that produce the fluctuations.

Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

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.