Statistical analysis of oligonucleotide microarray data

Microchip arrays have become one of the most rapidly growing techniques for monitoring gene expression at the genomic level and thereby gaining valuable insight about various important biological mechanisms. Examples of such mechanisms are: identifying disease-causing genes, genes involved in the regulation of some aspect of the cell cycle, etc. In this article, we discuss the problem of estimating gene expression based on a proper statistical model. More precisely, we show how the model introduced by Li and Wong can be used in its full bivariate generality to provide a new measure of gene expression from high-density oligonucleotide arrays. We also present a second gene expression index based on a new way of reducing the model into a simpler univariate model. In both cases, the gene expression indices are shown to be unbiased and to have lower variance than the established ones. Moreover, we present a bootstrap method aiming at providing non-parametric confidence intervals for the expression index.     

Babesia bovis: the development of an expression oligonucleotide microarray

The availability of a stage-specific Babesia bovis expression profile can facilitate the identification of candidate vaccine antigens. In addition, highly expressed genes during a particular developmental stage may suggest their relevance during that stage. In this study, we generated and validated a custom B. bovis high density oligonucleotide microarray that can be used to examine gene expression levels. An expression profile of in vitro cultured intraerythrocytic stage genes that could be distinguished from contaminating host message was established, and the expression levels of over 1000 genes were ranked. Ranking order was validated using quantitative real time PCR on a twelve randomly selected open reading frames whose expression levels range from the highest to the acceptable lowest. Expression of annotated ORFs was consistent with results from a recently published B. bovis expression sequence tag study. Therefore, we conclude that the microarray is suitable for analyzing B. bovis gene expression, and present the complete B. bovis infected erythrocyte expression profile.     

HOXD-AS1 is a predictor of clinical progression and functions as an oncogenic lncRNAs in papillary thyroid cancer

Long noncoding RNA HOXD cluster antisense RNA 1 (HOXD-AS1) has been found to play a crucial role in the tumorigenesis and progression of human cancer. However, the role of HOXD-AS1 in papillary thyroid cancer is still unknown. The purpose of this study was to investigate the clinical value and biological function of HOXD-AS1 in papillary thyroid cancer. The results showed HOXD-AS1 is overexpressed in papillary thyroid cancer tissues and cell lines compared with matching nontumor tissue specimens and normal thyroid cell line, respectively. High expression of HOXD-AS1 was associated with elderly people, advanced clinical stage, large tumor size, present lymph node metastasis, and distant metastasis. There was no significant correlation between HOXD-AS1 expression and disease-free survival or overall survival in this cohort from the TCGA database. The study in vitro suggested reduced HOXD-AS1 expression inhibited papillary thyroid cancer cell proliferation, migration, and invasion, and promoted cell-cycle arrest. In conclusion, HOXD-AS1 is a biomarker for predicting clinical progression in papillary thyroid cancer.     

Gene expression profiling in mitochondrial disease: assessment of microarray accuracy by high-throughput Q-PCR

Mitochondrial diseases are a heterogeneous array of disorders with a complex etiology. Use of microarrays as a tool to investigate complex human disease is increasingly common, however, a principle drawback of microarrays is their limited dynamic range, due to the poor quantification of weak signals. Although it is generally understood that low-intensity microarray 'spots' may be unreliable, there exists little documentation of their accuracy. Quantitative PCR (Q-PCR) is frequently used to validate microarray data, yet few Q-PCR validation studies have focused on the accuracy of low-intensity microarray signals. Hence, we have used Q-PCR to systematically assess microarray accuracy as a function of signal strength in a mouse model of mitochondrial disease, the superoxide dismutase 2 (SOD2) nullizygous mouse. We have focused on a unique category of data--spots with only one weak signal in a two-dye comparative hybridization--and show that such 'high-low' signal intensities are common for differentially expressed genes. This category of differential expression may be more important in mitochondrial disease in which there are often mosaic expression patterns due to the idiosyncratic distribution of mutant mtDNA in heteroplasmic individuals. Using RNA from the SOD2 mouse, we found that when spotted cDNA microarray data are filtered for quality (low variance between many technical replicates) and spot intensity (above a negative control threshold in both channels), there is an excellent quantitative concordance with Q-PCR (R2 = 0.94). The accuracy of gene expression ratios from low-intensity spots (R2 = 0.27) and 'high-low' spots (R2 = 0.32) is considerably lower. Our results should serve as guidelines for microarray interpretation and the selection of genes for validation in mitochondrial disorders.     

反义RNA的作用机理及其在植物基因工程领域的应用

本文综述了反义RNA在原核生物和真核生物中的作用机理,并系统地介绍了反义RNA技术在植物基因过程中的研究进展。     

In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis

BsrE/SR5 is a new type I toxin/antitoxin system located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3′ ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (k_app) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA system from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.     

Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

Assessment and integration of publicly available SAGE, cDNA microarray, and oligonucleotide microarray expression data for global coexpression analyses

Large amounts of gene expression data from several different technologies are becoming available to the scientific community. A common practice is to use these data to calculate global gene coexpression for validation or integration of other "omic" data. To assess the utility of publicly available datasets for this purpose we have analyzed Homo sapiens data from 1202 cDNA microarray experiments, 242 SAGE libraries, and 667 Affymetrix oligonucleotide microarray experiments. The three datasets compared demonstrate significant but low levels of global concordance (rc<0.11). Assessment against Gene Ontology (GO) revealed that all three platforms identify more coexpressed gene pairs with common biological processes than expected by chance. As the Pearson correlation for a gene pair increased it was more likely to be confirmed by GO. The Affymetrix dataset performed best individually with gene pairs of correlation 0.9-1.0 confirmed by GO in 74% of cases. However, in all cases, gene pairs confirmed by multiple platforms were more likely to be confirmed by GO. We show that combining results from different expression platforms increases reliability of coexpression. A comparison with other recently published coexpression studies found similar results in terms of performance against GO but with each method producing distinctly different gene pair lists.     

Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR

Aims:  Breast milk has been described as a source of bacteria influencing the development of the infant gut microbiota. Up to the present, few studies have been focused on the application of culture-independent techniques to study bacterial diversity in breast milk. In this context, the aim of this study was to characterize the breast milk microbiota of healthy women by applying the quantitative real-time PCR technique (qRTi-PCR).
Methods and Results:  A total of 50 breast milk samples were analysed by qPCR to assess the presence of different bacterial genera or clusters, including the Bifidobacterium , Lactobacillus , Staphylococcus , Bacteroides , Enterococcus , Streptococcus , Clostridium cluster IV and Clostridium cluster XIVa–XIVb groups. Staphylococcus , Streptococcus , Bifidobacterium and Lactobacillus were the predominant groups and were detected in all the samples. Clostridium XIVa–XIVb and Enterococcus were detected in most of the samples in contrast to the Bacteroides and Clostridium cluster IV groups.
Conclusions:  Our results confirm the abundance of bacterial DNA in breast milk samples and suggest that the qRTi-PCR technique has a huge potential in the microbiological analysis of human milk.
Significance and Impact of the study:  qRTi-PCR allowed the detection of bacterial DNA of streptococci, staphylococci, lactic acid bacteria and bifidobacteria in the samples of human milk, which confirms that breast milk can be an important source of bacteria and bacterial DNA to the infant gut.     

三种不同方法固定的石蜡切片中RNA的分析

研究在 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定的石蜡切片中提取RNA的质量和数量 .取 2 5 0g体重的Wistar大鼠的肾脏 ,分别采用 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定 ,石蜡包埋 ,H E染色 ;采用RNA裂解液、TRIZOL试剂 2种方法提取切片RNA ,逆转录为cDNA ,采用普通PCR和SYBRGREEN 1定量PCR分析RNA质量和数量 .结果表明 ,3种固定方法都可保持组织良好的结构和形态 ;采用 2种提取方法 ,均可经RT PCR扩增出 180bp大鼠磷酸甘油醛脱氢酶 (G3PDH)、5 6 5bpβ肌动蛋白 (β actin)、10 0bp纤溶酶系活化剂抑制物 1(PAI 1) ;但采用RNA裂解液时 ,比TRIZOL试剂可提取更多的RNA .     

猪组织中miR-103实时定量PCR分析时合适内参的确定

定量实时PCR是miRNA表达检测的主要方法之一,而利用合适内参对定量实时PCR数据进行校正处理是确保该方法分析准确性的关键.为了确定猪正常组织中miR-103定量实时PCR检测分析的合适内参,首先采用geNorm算法和扩增效率试验对miR-196、U6 snRNA和总RNA 3个候选内参进行了评价;然后以miR-103的绝对定量结果为对照,比较分析了3个候选内参的校正准确性．结果表明,miR-196的表达稳定性和扩增效率均优于U6 snRNA和总RNA;以miR-196为内参的miR-103相对定量结果同绝对定量结果具有较高的一致性,两者均显示miR-103在猪大脑中高丰度表达,在胃、小脑、小肠、心脏、肝脏和胰脏中适度表达,在肺、脾脏和腿肌中轻度表达.这些结果说明,miR-196可作为猪正常组织中miR-103定量实时PCR相对定量分析的一个合适内参.