Validation of rat reference genes for improved quantitative gene expression analysis using low density arrays

Real-time PCR has become increasingly important in gene expression profiling research, and it is widely agreed that normalized data are required for accurate estimates of messenger RNA (mRNA) expression. With increased gene expression profiling in preclinical research and toxicogenomics, a need for reference genes in the rat has emerged, and the studies in this area have not yet been thoroughly evaluated. The purpose of our study was to evaluate a panel of rat reference genes for variation of gene expression in different tissue types. We selected 48 known target genes based on their putative invariability. The gene expression of all targets was examined in 11 types of rat tissues using TaqMan low density array (LDA) technology. The variability of each gene was assessed using a two-step statistical model. The analysis of mean expression using multiple reference genes was shown to provide accurate and reliable normalized expression data. The least five variable genes from each specific tissue were recommended for future tissue-specific studies. Finally, a subset of investigated rat reference genes showing the least variation is recommended for further evaluation using the LDA platform. Our work should considerably enhance a researcher's ability to simply and efficiently identify appropriate reference genes for given experiments.     

Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans

To identify reliable reference genes for toxicological studies, 16 commonly-used reference genes were selected as candidates to evaluate their expression stabilities under experimental conditions in Caenorhabditis elegans. Sixteen candidates were composed of 12 protein-coding genes and 4 non-coding RNAs, they were act-2, ama-1, arp-6, cdc-42, csq-1, eif-3.C, idhg-1, mdh, pmp-3, rbd-1, tba-1, Y45F10D.4, 18S rRNA, Ce234, U18, and U6. Larval stage 1 synchronized hermaphrodites were exposed to benzo-α-pyrene (BαP), chlorpyrifos, diazinon, gossypol, zinc oxide nanoparticles, and the vehicle control DMSO for 30 h, respectively. Expression stabilities of candidate genes were analyzed using 4 independent evaluating approaches (BestKeeper, the delta Ct approach, geNorm, and NormFinder) followed by a comprehensive method. Results showed that there were slight differences in ranking order between evaluation methods due to their different assumptions and computations. The results also showed that responses of candidate genes to different chemicals were distinct, 18S rRNA was the best for BαP and chlorpyrifos, tba-1 was the most stable gene for diazinon and gossypol treatments, while pmp-3 was more stable for zinc oxide exposure. Additionally, results demonstrated that combinations of multiple genes were more reliable than individual gene, suggesting selecting two or more candidates as reference genes may generate more reliable results for toxicological studies.     

miRNAs调控lincRNAs的生物信息学预测与功能分析

基因间长链非编码RNAs(Long intergenic non-coding RNAs, lincRNAs)是位于蛋白编码基因之间的长度超过200 nt的非编码RNAs, 在动物中参与细胞周期调控、免疫监视、胚胎干细胞分化等多种生物学过程, 但是lincRNAs在大多数植物中的功能尚不清楚。MicroRNAs(miRNAs)是真核生物中一类在转录水平和转录后水平介导基因沉默的21 nt左右的内源性单链小非编码RNAs分子, 通过序列互补的方式调控靶标基因的表达。目前miRNAs的靶标研究主要集中于编码蛋白的基因, 而对于靶标为非编码RNAs的研究较少, 尤其在植物中的研究更为少见。为了系统挖掘植物中lincRNAs的功能, 文章整合miRNAs数据、cDNAs数据和降解组数据, 利用生物信息学方法找到拟南芥(Arabidopsis thaliana)337个成熟miRNAs在2708个lincRNAs上的可能结合位点, 构建了miRNAs-mRNAs-lincRNAs调控网络, 并根据竞争性内源(ceRNA)假说预测lincRNAs的功能, 为进一步阐明植物中miRNAs对lincRNAs的调控机制以及lincRNAs的功能奠定了基础。     

Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease

More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.     

Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease

More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.     

长非编码RNA在基因调控和肿瘤形成中的作用

哺乳动物中,只有小部分基因转录成为编码蛋白质的RNA,大量的基因则转录为不能编码蛋白质的RNA,即ncRNA。长非 编码RNA(lncRNAs)是分子长度在200-100000 nt 之间的一类ncRNA。lncRNAs 的数量超过蛋白质编码基因的数量。目前,对长非 编码RNA(lncRNAs)的生物学特性,转录调控以及其在肿瘤发生发展中的作用机制的研究任然是RNA研究的热点。lncRNAs 通 过控制染色质重塑,转录调控和录转录后调控而在基因的转录调节中发挥了重要作用。lncRNAs 与多种肿瘤相关,并且在抑制因 素和促进因素中都具有重要的作用。众多文献报道的结果表明lncRNAs 参与调控基因表达,在正常细胞与肿瘤细胞的转换中起 到至关重要的作用。     

Identification and characterization of small RNAs from vernalized<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two major classes of small non-coding RNAs with important roles in the regulation of gene expression, such as mRNA degradation and translational repression, heterochromatin formation, genome defense against transposons and viruses in eukaryotes. MiRNA- and siRNA-directed processes have emerged as a regulatory mechanism for growth and development in both animals and plants. To identify small RNAs that might be involved in vernalization, a process accelerating flowering brought on by a long period of cold, we generated a library of small RNAs from Arabidopsis that had been subject to vernalization. From the analysis of the library, 277 small RNAs were identified. They were distributed throughout all the five chromosomes. While the vast majority of small RNA genes locate on intergenic regions, others locate on repeat-rich regions, centromeric regions, transposon-related genes, and protein-coding genes. Five of them were mapped to convergent overlapping gene pairs. Two-hundred and forty of them were novel endogenous small RNAs that have not been cloned yet from plants grown under normal conditions and other environmental stresses. Seven putative miRNAs were up- or down-regulated by vernalization. In conclusion, many small RNAs were identified from vernalized Arabidopsis and some of these identified small RNAs may play roles in plant responses to vernalization.     

Evaluation of the reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the green peach aphid,Myzus persicae

The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT‐PCR has been used for target gene expression analysis on M. persicae. Using real time qRT‐PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔC_t method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT‐PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research.     

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.     

Complete mitochondrial genome of a Chinese scorpion Mesobuthus martensii (Chelicerata, Scorpiones, Buthidae).

The complete mitochondrial genome (15,034 bp) of a Chinese scorpion Mesobuthus martensii (Buthidae) was sequenced and characterized in detail. The genome contains 13 protein-coding genes, 21 transfer RNA genes, two ribosomal RNA genes and a large non-coding region ( = CR). Its gene arrangement pattern is identical to that of Limulus polyphemus (Chelicerata, Xiphosura), with the exceptions of the tRNA(Glu)-tRNA(Ile)-tRNA(Met) (Q-I-M) arrangement and tRNA(Asp)-loss. Additional interesting features are found and discussed: high frequency of Leu(UUG) codon use, low A+T content of the genome (66.75%), and six repeat units (five 60-nt-long and one 58-nt-long repeats) in the 998-nt CR. Bayesian analysis based on amino acid sequences of the 12 proteincoding genes (excluding ATP8) reveals that the family Buthidae (Order Scorpiones) and the class Arachnida form strong monophyletic groups within Chelicerata, respectively. It indicated that the scorpions are the most ancestral arachnids.     

Human Disease-Associated Genetic Variation Impacts Large Intergenic Non-Coding RNA Expression

Recently it has become clear that only a small percentage (7%) of disease-associated single nucleotide polymorphisms (SNPs) are located in protein-coding regions, while the remaining 93% are located in gene regulatory regions or in intergenic regions. Thus, the understanding of how genetic variations control the expression of non-coding RNAs (in a tissue-dependent manner) has far-reaching implications. We tested the association of SNPs with expression levels (eQTLs) of large intergenic non-coding RNAs (lincRNAs), using genome-wide gene expression and genotype data from five different tissues. We identified 112 cis-regulated lincRNAs, of which 45% could be replicated in an independent dataset. We observed that 75% of the SNPs affecting lincRNA expression (lincRNA cis-eQTLs) were specific to lincRNA alone and did not affect the expression of neighboring protein-coding genes. We show that this specific genotype-lincRNA expression correlation is tissue-dependent and that many of these lincRNA cis-eQTL SNPs are also associated with complex traits and diseases.