Single-cell molecular-biology is a relatively new scientific branch in biology. The first single-cell analysis were involved in the characterization of mitochondrial DNA in 1988. Single-cell DNA analysis, in particular genomic DNA, is important and may be informative in the analysis of genetics of cell clonality, genetic anticipation and single-cell DNA polymorphisms. Nowadays for most scientists the quantitative transcriptomics in a single-cell is much more important, and the analytical method of choice is the quantitative real-time RT-PCR. In single-cell biology the absolute abundance of particular mRNAs or microRNAs and their up- or down-regulation in a single cell, compared to their neighbour cells, is the goal. The need for quantitative single-cell mRNA analysis is evident given the vast cellular heterogeneity of all tissue cells and the inability of conventional RNA methods, like northern blotting, RNAse protection assay or classical block RT-PCR, to distinguish individual cellular contributions to mRNA abundance differences.

The purpose of this single-cell qPCR page is to provide researchers with resources (papers, talks, posters) for single-cell molecular analysis such as qPCR and RT-qPCR:

• Research Highlight:  Gene expression - Which mean do you mean ?  Bengtsson et al. 2005
  
• Single-cell handling subdomain
  
• Technique:  Micro-manipulation techniques
• Technique:  Laser Capture Microdissection
• Technique:  Single cell sorting and cloning
  
• Laser-Capture Microdissection  and  qRT-PCR
• Extraction:  A new DNA extraction method for efficient single-cell genetic diagnosis
  
• Extraction:  QuantiLyse - Reliable DNA Amplification from Single Cells
• Technique:  LATE-PCR - Linear-After-The-Exponential (LATE)-PCR
• Thesis:  SINGLE-CELL GENE-EXPRESSION ANALYSIS BY QUANTITATIVE RT-PCR  (by Luigi Warren)
• Poster: TaqMan PreAmp Master Mix Kit for Real-Time Gene Expression Analysis with Sample Limited Specimens.
• Single-cell qPCR application papers:    page 2    page 3   page 4   page 5   &  page 6  with newest update from June 2014

		Single-Cell -omics in Nature Reviews Recent technological advances are providing unprecedented opportunities to analyse the complexities of biological systems at the single-cell level. Various crucial biological phenomena are either invisible or only partially characterized when interrogated using standard analyses that average data across a bulk population of cells. However, high-throughput analyses of the genomes, transcriptomes and proteomes of single cells are providing novel and important insights into diverse processes such as development, gene-expression dynamics, tissue heterogeneity and disease pathogenesis.
		Sequencing the Single Cell – Adventures in Genomics by Illumina Inc. A single cell is the smallest building block in biology. Each and every cell contains an entire genome with all the information to create an entire organism – be it a bacterium or a buffalo cell. Recent advances in sequencing technology are making it possible to extract and sequence the genomes from individual cells. This is advancing our understanding of many biological processes. https://www.youtube.com/user/IlluminaInc http://www.illumina.com/science/education/adventures-in-genomics.html

Expression profiling of single mammalian cells - small is beautiful.
Brady G.
School of Biological Sciences, G.38 Stopford Building, University of Manchester,
Oxford Road, Manchester M13 9PT, UK.
Yeast. 2000 Sep 30;17(3):211-217

Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.

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Current applications of single-cell PCR.
Hahn S, Zhong XY, Troeger C, Burgemeister R, Gloning K, Holzgreve W.
Department of Obstetrics and Gynaecology, University of Basel, Switzerland
Cell Mol Life Sci. 2000 57(1): 96-105

The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.

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Research Highlight  -  Gene expression: Which mean do you mean?
Patrick Goymer
Nature Reviews Genetics 6, 877 (December 2005)
http://www.nature.com/nrg/journal/v6/n12/full/nrg1758.html

There is considerable variation in gene-expression levels between individual cells. Bengtsson et al. show that these levels are distributed log-normally rather than normally, which implies that the arithmetic mean does not represent the situation in a typical cell. They also show that the levels of expression of different genes in the same cell do not generally correlate, and suggest that mechanistic conclusions can be drawn when they do. Using reverse transcriptase quantitative real-time PCR, they measured the transcript levels of 5 genes in 169 mouse pancreatic cells. For each gene the results were distributed log-normally across the sample cells, making the geometric mean a more appropriate representation of the data than the more commonly quoted arithmetic mean. For the insulin genes, Ins1 and Ins2, up to 9-fold differences were found between the arithmetic and geometric means. Of the five genes studied, only Ins1 and Ins2 expression levels correlated at the level of the individual cell. Levels of ActB, the -actin gene, correlated with these two only at the overall population level, whereas levels of the final two genes did not correlate with any of the others. This indicates that expression-level differences in individual genes are not due to cells having different levels of overall transcription. The authors suggest that genes that correlate at the individual cell level are co-ordinately regulated, whereas those that correlate at the population level merely respond to the same environmental stimuli. The importance of these findings is demonstrated by the fact that we might have underestimated the effect of glucose on insulin expression by almost 4-fold, which could be important in the administration of therapeutic insulin.

References (see below)
Bengtsson, M. et al. Gene-expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Res. 15, 1388–1392 (2005)

Global cDNA amplification combined with real-time RT-PCR: 
accurate quantification of multiple human potassium channel genes at the single cell level.
Al-Taher A, Bashein A, Nolan T, Hollingsworth M, Brady G. (2000)
Yeast. 2000 Sep 30;17(3): 201-210

We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitativeRT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, isable to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst 10(8) total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

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Correlating function and gene expression of individual basal ganglia neurons.
 Liss B, Roeper J.
Molecular Neurobiology, Institute for Physiology, Philipps-University Marburg, Deutschhausstrasse 2, 35033 Marburg, Germany.
Trends Neurosci. 2004 Aug;27(8):475-81

Functional studies at the level of individual neurons have greatly contributed to our current understanding of basal ganglia function and dysfunction. However, identification of the expressed genes responsible for these distinct neuronal phenotypes is less advanced. Qualitative and quantitative single-cell gene-expression profiling, combined with electrophysiological analysis, allows phenotype-genotype correlations to be made for individual neurons. In this review, progress on gene-expression profiling of individual, functionally characterized basal ganglia neurons is discussed, focusing on ion channels and receptors. In addition, methodological issues are discussed and emerging novel techniques are introduced that will enable a genome-wide comparison of function and gene expression for individual neurons.

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University Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit,
Department of Pharmacology, Oxford University, Parks Road, Oxford OX1 3PT, UK.

The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that  the amplification kinetics of rtqPCRs were dramatically altered in a  non-systematic fashion. Here, I describe a simple and robust precipitation  protocol suitable for purification of single-cell cDNA that completely removes  inhibitory RT components without detectable loss of cDNA. This improved  single-cell real-time RT-PCR protocol provides a powerful tool to quantify  differential gene expression of individual cells and thus could complement  global microarray-based expression profiling strategies.

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Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.
Hartshorn C, Anshelevich A, Wangh LJ.
BMC Biotechnol. 2005 5(1): 2.

Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost. This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres. This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.

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Real-time PCR with molecular beacons provides a highly accurate assay for detection of Tay-Sachs alleles in single cells.
Rice JE, Sanchez JA, Pierce KE, Wangh LJ.
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.
Prenat Diagn. 2002 Dec;22(12): 1130-1134

The results presented here provide the first single-cell genetic assay for Tay-Sachs disease based on real-time PCR. Individual lymphoblasts were lysed with an optimized lysis buffer and assayed using one pair of primers that amplifies both the wild type  and 1278 + TATC Tay-Sachs alleles. The resulting amplicons were detected in real time with two molecular beacons each with a different colored fluorochrome. The kinetics of amplicon accumulation generate objective criteria by which to evaluate the validity of each reaction. The assay had an overall utility of 95%, based on the detection of at least one signal in 235 of the 248 attempted tests and an efficiency of 97%, as 7 of the 235 samples were excluded from further analysis for objective quantitative reasons. The accuracy of the assay was 99.1%, because 228 of 230 samples gave signals consistent with the genotype  of the cells. Only two of the 135 heterozygous samples were allele drop-outs, a rate far lower than previously reported for single-cell Tay-Sachs assays  using conventional methods of PCR.

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Real-time quantitative RT-PCR after laser-assisted cell picking
Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM.
Department of Pathology, Justus-Liebig-Universitat Giessen, Germany.
Nat Med. 1998 4(11): 1329-1333.

The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifoldupregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.

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There remains uncertainty and some controversy about the percentages and types of cells in human sensory nerve ganglia that harbor latent herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) DNA. We developed and validated laser-capture microdissection and real-time PCR (LCM/PCR) assays for the presence and copy numbers of HSV-1 gG and VZV gene 62 sequences in single cells recovered from sections of human trigeminal ganglia (TG) obtained at autopsy. Among 970 individual sensory neurons from five subjects, 2.0 to 10.5% were positive for HSV-1 DNA, with a median of 11.3 copies/positive cell, compared with 0.2 to 1.5% of neurons found to be positive by in situ hybridization (ISH) for HSV-1 latency-associated transcripts (LAT), the classical surrogate marker for HSV latency. This indicates a more pervasive latent HSV-1 infection of human TG neurons than originally thought. Combined ISH/LCM/PCR assays revealed that the majority of the latently infected neurons do not accumulate LAT to detectable levels. We detected VZV DNA in 1.0 to 6.9% of individual neurons from 10 subjects. Of the total 1,722 neurons tested, 4.1% were VZV DNA positive, with a median of 6.9 viral genomes/positive cell. After removal by LCM of all visible neurons on a slide, all surrounding nonneuronal cells were harvested and assayed: 21 copies of HSV-1 DNA were detected in 5,200 nonneuronal cells, while nine VZV genomes were detected in 14,200 nonneuronal cells. These data indicate that both HSV-1 and VZV DNAs persist in human TG primarily, if not exclusively, in a moderate percentage of neuronal cells.

Analysis of connective tissues by laser capture microdissection
and reverse transcriptase-polymerase chain reaction
Robin Jacquet, Jennifer Hillyer, William J. Landis*
Department of Biochemistry and Molecular Pathology
Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA

Studies of gene expression from bone, cartilage, and other tissues are complicated by the fact that their RNA, collected and pooled for analysis, often represents a wide variety of composite cells distinct in individual phenotype, age, and state of maturation. Laser capture microdissection (LCM) is a technique that allows specific cells to be isolated according to their phenotype, condition, or other marker from within such heterogeneity. As a result, this approach can yield RNA that is particular to a subset of cells comprising the total cell population of the tissue. This study reports the application of LCM to the gene expression analysis of the cartilaginous epiphyseal growth plate of normal newborn mice. The methodology utilized for this purpose has been coupled with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) to quantitate the expression of certain genes involved in growth plate development and calcification. In this paper, the approaches used for isolating and purifying RNA from phenotypically specific chondrocyte populations of the murine growth plate are detailed and illustrate and compare both qualitative and quantitative RT-PCR results. The technique will hopefully serve as a guide for the further analysis of this and other connective tissues by LCM and RT-PCR.

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IFIHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, timeconsuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.

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