We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

/pdf/direct-multiplexed-measurement-of-gene-expression-with-color-48nvma70wx.pdf

Direct multiplexed measurement of gene expression with color-coded probe pairs.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2009.10.036

Correlation of mRNA and protein in complex biological samples

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

/pdf/genome-wide-survey-of-human-alternative-pre-mrna-splicing-1nzek80y6m.pdf

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

Recent genome-wide analyses of alternative splicing indicate that 40-60% of human genes have alternative splice forms, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Here we review these recent results from bioinformatics studies, assess their reliability and consider the impact of alternative splicing on biological functions. Although the 'big picture' of alternative splicing that is emerging from genomics is exciting, there are many challenges. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. We recommend a community-based effort to discover and characterize alternative splice forms comprehensively throughout the human genome.

A genomic view of alternative splicing

The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977; Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999; Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002).

http://genesdev.cshlp.org/content/17/4/419.full.pdf

Pre-mRNA splicing and human disease

To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of ∼10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any ‘non-target’ transcripts (cDNAs) >75% similar over the 50 base target may show crosshybridization. Thus non-target sequences which have >75–80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity. In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results validate the utility of oligonucleotide probe (50mer) glass microarrays.

/pdf/assessment-of-the-sensitivity-and-specificity-of-2221cwe28n.pdf

Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays

Alternative splicing of premessenger RNA is an important layer of regulation in eukaryotic gene expression. Splice variation of a large number of genes has been implicated in various cell growth and differentiation processes. To measure tissue-specific splicing of genes on a large scale, we collected gene expression data from 11 rat tissues using a high-density oligonucleotide array representing 1600 rat genes. Expression of each gene on the chip is measured by 20 pairs of independent oligonucleotide probes. Two algorithms have been developed to normalize and compare the chip hybridization signals among different tissues at individual oligonucleotide probe level. Oligonucleotide probes (the perfect match [PM] probe of each probe pair), detecting potential tissue-specific splice variants, were identified by the algorithms. The identified candidate splice variants have been compared to the alternatively spliced transcripts predicted by an EST clustering program. In addition, 50% of the top candidates predicted by the algorithms were confirmed by RT-PCR experiment. The study indicates that oligonucleotide probe-based DNA chip assays provide a powerful approach to detect splice variants at genome scale.

/pdf/predicting-splice-variant-from-dna-chip-expression-data-3um8j6qgsp.pdf

Predicting Splice Variant from DNA Chip Expression Data

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation.

Steven J Madore

Papers

Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays

Predicting Splice Variant from DNA Chip Expression Data

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation.