2′-MeO-RNA Containing Oligodeoxyribonucleotide Primers Can Prevent Template-independent Base Extension on Microarrays

DNA microarrays require tens of thousands of deoxyoligonucleotides to be registered in an addressable fashion through immobilization, so that they have the high-throughput capability of analyzing a large number of samples simultaneously in a minimal volume of each reagent. However, using immobilized DNA molecules on microarrays can impose certain technical problems for some assays. For example, high background noise has been observed in using immobilized oligonucleotide microarrays (DNA chip) for primer extension reactions. This noise may be associated with the reactions of secondary structures formed by the adjacent primers physically constrained on the surface. Single-base extension (SBE) of arrayed primers on a chip has been extensively used in mini-sequencing to examine single nucleotide polymorphisms (SNP). Some primers appeared to be extendable in the absence of any template and thus competed against the base extension directed by the assay target such as genomic DNA. In this article, a method is reported that is capable of reducing template-independent extension by the substitution of a 2′-methoxyribonucleotide in the otherwise oligodeoxyribonucleotide primer. The surrogate compound placed at the 5′-end of the putative secondary structure sequence of a given primer was able to inhibit template-independent extension and to improve data quality of surface-attached primer extension assays.     

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.     

Muscle Gene Expression Associated with Increased Marbling in Beef Cattle

The objective of this study was to identify specific bovine genes expressed within skeletal muscle that are associated with intramuscular fat deposition. Twenty-eight Angus-Simmental cross steers and heifers were harvested at the University of Illinois Meat Science Laboratory. Four pairs of animals were identified based on similar adjusted backfat thickness but differing amounts of intramuscular fat within each pair. RNA was extracted from muscle samples devoid of visible fat and microarray analysis was performed. Based on this analysis, 9 genes were selected and expression was subsequently confirmed by qPCR. Expression levels of MYH3, HOXD10, MXRA8, and CASQ2 were increased in animals with high marbling, whereas levels of NPNT, MRC1, DNER, and CYPB4 were decreased in high marbled animals. The remaining gene, ACTN2 was determined to be a false positive and was, therefore, excluded from further study. Despite the positive results of the preliminary study, associations between gene expression and intramuscular fat content did not extend to the larger population of cattle. A significant negative association existed between expression of MRC1 and marbling level (P = 0.04). Therefore, this study was unable to identify a particular skeletal muscle gene set whose expression correlated well with marbling levels in the larger population of beef cattle.     

The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation,migration and invasion

Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease.

Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G₁ cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced.

Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis.     

结直肠癌组织异常表达miRNA的鉴定

目的：筛选结直肠癌组织异常表达的miRNAs。方法：采用Agilem基因芯片（V12．0）分析结直肠癌组织及其配对正常粘膜组织间差异表达的miRNAs,MiRNAs错误发生率（FDR）〈0．05和微矩阵显著性分析（SAM）q值〈0．05为差异显著。结果：鉴定出结直肠癌中32个差异表达的miRNAs,显著上调和下调各16个。实时定量PCR（RT—qPCR）证实基因芯片中4个表达上调的miRNAs在结直肠癌组织中也显著上调。结论：MiRNA基因芯片鉴定出了结直肠癌组织一系列新的差异表达的miRNAs。