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Several molecular methods like real-time PCR (Q-PCR), expression sequence tag (EST) scan, microarray and microRNA analysis, and massively parallel signature sequencing (MPSS) have proved to be increasingly sensitive and ef fi cient for monitoring human embryonic stem cell (hESC) differentiation. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a need of rapid, cost-effective,robust, yet sensitive method for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of germ-layer-speci fi c gene markers. To determine the modulation of gene expression in hESCs during propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and de fi nitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising of set 1; and 3 lineage-speci fi c markers from each ecto-, meso-, and endoderm layers were combined as sets 2, 3, and 4,respectively. In summary, this study was performed to characterize hESCs on a molecular level and to determine the quality and degree of variability among hESC and their early progenies (EB). This singlereaction mxPCR assay was fl exible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation

Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.

Massively parallel signature sequencing (MPSS) is a technology capable of sequencing simultaneously almost all the DNA molecules in a sample. This technology is well suited for deep profiling of mRNA and small RNA by the sequencing of cDNA tags. A series of mRNA MPSS databases has been created from various libraries derived from four different species (Arabidopsis, rice, grape, and Magnaporthe grisea, the rice blast fungus). Our mRNA MPSS databases measure the absolute expression level of most genes in the sample and provide information about potentially novel transcripts (antisense transcripts, alternative splice isoforms, and regulatory intergenic transcripts). In addition to these data, we have recently built an extensive database from MPSS-derived Arabidopsis small RNA samples. All the databases are accessible thorough our Web interface (http://mpss.udel.edu), and the individual pages are equipped with various graphical and analytical tools. Here, we focus on a subset of these tools (e.g., Gene Analysis [GA], Chromosome Viewer [CV], and Library Analysis [LIBAN]) and describe how the users can analyze and interpret our MPSS expression data.

Massively parallel signature sequencing is a sequencing-based method that provides quantitative gene expression data for nearly all transcripts in a particular ribonucleic acid sample. Although the sequencing technology is practiced as a service by a California-based company, we have developed methods for the handling and analysis of these data. This chapter describes the steps involved in obtaining data from massively parallel signature sequencing, aligning the signatures to genomic sequence, identifying novel transcripts, and performing quantitative analyses of genes expressed under conditions such as disease treatments.

Massively parallel signature sequencing is an ultra-high throughput sequencing technology. It can simultaneously sequence millions of sequence tags, and, therefore, is ideal for whole genome analysis. When applied to expression profiling, it reveals almost every transcript in the sample and provides its accurate expression level. This chapter describes the technology and its application in establishing stem cell transcriptome databases.

Serial analysis of gene expression (SAGE™) is a patented, large-scale mRNA-profiling technology that produces comprehensive, quantitative, and reproducible gene expression profiles (originally described in refs. 1 and 2). Unlike the alternative technologies of differential display and subtractive hybridization, SAGE produces a full catalog of all transcripts, not only differentially expressed genes, and unlike smaller arrays, SAGE needs no assumptions about the genes that are likely to be expressed, thus allowing the identification of novel genes (are among many excellent reviews, see refs. 3, 4, 5). SAGE is based on generating clones of concatenated (linked) short sequence tags derived from mRNA from the target cells or tissue (Fig. 1).