A decade of improvements in quantification of gene expression and internal standard selection

Major improvements have been made in mRNA quantification and internal standard selection over the last decade. Our aim in this paper is to present the main developments that are of interest for practical laboratory work, contrasting the situation as it is now with the one of ten years ago, and presenting some excellent examples of what can be done today. Specifically, we will mainly discuss Real-Time RT-PCR major improvements that have been performed in the following areas: the most commonly used quantification techniques, the mathematical and software tools created to help researchers in their work on internal standard selection, the availability of detection chemistries and technical information and of commercial tools and services. In addition to mRNA quantification, we will also discuss some aspects of non-coding RNA and protein quantification. In addition to technical improvements, the development of international cooperation and the creation of technical databases are likely to represent a major tool for the future in the standardization of gene expression quantification.     

A quantification model for apoptosis in mouse embryos in the early stage of fetation

Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage, which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.     

GenOrigin: A comprehensive protein-coding gene origination database on the evolutionary timescale of life

The origination of new genes contributes to the biological diversity of life. New genes may quickly build their network, exert important functions, and generate novel phenotypes. Dating gene age and inferring the origination mechanisms of new genes, like primate-specific genes, is the basis for the functional study of the genes. However, no comprehensive resource of gene age estimates across species is available. Here, we systematically date the age of 9,102,113 protein-coding genes from 565 species in the Ensembl and Ensembl Genomes databases, including 82 bacteria, 57 protists, 134 fungi, 58 plants, 56 metazoa, and 178 vertebrates, using a protein-family-based pipeline with Wagner parsimony algorithm. We also collect gene age estimate data from other studies and uniformly distribute the gene age estimates to time ranges in a million years for comparison across studies. All the data are cataloged into GenOrigin (http://genorigin.chenzxlab.cn/), a user-friendly new database of gene age estimates, where users can browse gene age estimates by species, age, and gene ontology. In GenOrigin, the information such as gene age estimates, annotation, gene ontology, ortholog, and paralog, as well as detailed gene presence/absence views for gene age inference based on the species tree with evolutionary timescale, is provided to researchers for exploring gene functions.     

A diffusion model for the fate of tandem gene duplicates in diploids

Suppose one chromosome in one member of a population somehow acquires a duplicate copy of the gene, fully linked to the original gene's locus. Preservation is the event that eventually every chromosome in the population is a descendant of the one which initially carried the duplicate. For a haploid population in which the absence of all copies of the gene is lethal, the probability of preservation has recently been estimated via a diffusion approximation. That approximation is shown to carry over to the case of diploids and arbitrary strong selection against the absence of the gene. The techniques used lead to some new results. In the large population limit, it is shown that the relative probability that descendants of a small number of individuals carrying multiple copies of the gene fix in the population is proportional to the number of copies carried. The probability of preservation is approximated when chromosomes carrying two copies of the gene are subject to additional, fully non-functionalizing mutations, thereby modelling either an additional cost of replicating a longer genome, or a partial duplication of the gene. In the latter case the preservation probability depends only on the mutation rate to null for the duplicated portion of the gene.     

基因芯片的功能评价与辅助设计软件GeneHubD

为了比较特定的基因芯片的功能偏岐 ,我们研制开发了一个简单实用的基因芯片功能评价与辅助设计软件GeneHubD。通过结合GO、KEGG、BIND、MIPS、Unists数据库的基因功能注释信息 ,该软件有助于实验人员选择或设计合适的芯片开展芯片实验     

Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq

Background

Massively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples.

Results

False-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values.

Conclusions

Our results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1767-y) contains supplementary material, which is available to authorized users.     

A Brassica campestris-alboglabra addition line and its use for gene mapping,intergenomic gene transfer and generation of trisomics

Summary Brassica campestris-alboglabra monosomic addition lines were developed from a trigenomic Brassica hybrid (2 n=3 x=29, AAC) obtained by backcrossing a resynthesized B. napus (2 n=4 x=38, AACC) line to its parental B. campestris (2 n=2 x=20, AA) line. One addition line was characterized genetically with three loci specific for the alien chromosome and cytologically by meiotic analysis. The following results were obtained. (1) The same chromosome in the B. alboglabra (2 n= 2 x=18, CC) genome carried the three loci, E _c, W _c and Lap-1 C ^c, which control the biosynthesis of erucic acid, white flower colour and the faster migrating band of leucine aminopeptidase, respectively. The linear order and possible positions of the three loci were inferred. The meiotic behaviour of the alien chromosome was documented and its transmission frequency was assessed. (2) Intergenomic recombination frequently occurred in the monosomic addition line, resulting in the introgression of one or two loci from the alien chromosome into the B. campestris genome. (3) B. campestris trisomics were found in the progeny of the monosomic addition line. (4) The removal of the other eight C-genome chromosomes from the trigenomic Brassica hybrid led to a dramatic increase in the erucic acid content of the monosomic addition line. (5) No offspring of the trigenomic Brassica hybrid showed evidence of intergenomic recombination and introgression of the W _c locus into the B. campestris genome. It is questioned whether such a difference might be due to a possible regulating mechanism for homoeologous chromosome pairing.     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.     

Carriage, quantification, and predominance of methanogens and sulfate-reducing bacteria in faecal samples

AIMS: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms. METHODS AND RESULTS: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults. CONCLUSIONS: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.     

Analysis of human disease genes in the context of gene essentiality

The characteristics of human disease genes were investigated through a comparative analysis with mouse mutant phenotype data. Mouse orthologs with mutations that resulted in discernible phenotypes were separated from mutations with no phenotypic defect, listing ‘phenotype’ and ‘no phenotype’ genes. First, we showed that phenotype genes are more likely to be disease genes compared to no phenotype genes. Phenotype genes were further divided into ‘embryonic lethal’, ‘postnatal lethal’, and ‘non-lethal phenotype’ groups. Interestingly, embryonic lethal genes, the most essential genes in mouse, were less likely to be disease genes than postnatal lethal genes. These findings indicate that some extremely essential genes are less likely to be disease genes, although human disease genes tend to display characteristics of essential genes. We also showed that, in lethal groups, non-disease genes tend to evolve slower than disease genes indicating a strong purifying selection on non-disease genes in this group. In addition, phenotype and no phenotype groups showed differing types of disease mutations. Disease genes in the no phenotype group displayed a higher frequency of regulatory mutations while those in the phenotype group had more frequent coding mutations, indicating that the types of disease mutations vary depending on gene essentiality. Furthermore, missense disease mutations in no phenotype genes were found to be more radical amino acid substitutions than those in phenotype genes.