Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at ?80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.     

大鼠血管组织血管紧张素原基因表达的实时PCR定量分析

目的:探讨大鼠血管组织血管紧张素原(angiotensinogen,AGT)低丰度mRNA表达的实时PCR定量分析方法,并将其用于检测模拟失重大鼠基底和股动脉血管组织AGT mRNA的表达.方法:提取8周模拟失重(SUS)与对照组(CON)大鼠血管组织的总RNA,进行反转录后,对目的基因AGT与内参照基因GAPDH的mRNA进行实时PCR分析.应用TaqMan-MGB探针,测出上述mRNA实时PCR反应的放大效率(E)及阈循环数Ct,再依据一定数学模型由E与Ct得出经GAPDH归一化的AGT mRNA表达变化.结果:与CON相比,SUS大鼠基底动脉组织AGT mRNA表达增加240%,而股动脉组织则降低66%.结论:本工作为定量检测大鼠血管组织低丰度mRNA表达提示了一种特异、灵敏、精确、重复性好的简便方法.     

Use of RNA and genomic DNA references for inferred comparisons in DNA microarray analyses

In most microarray assays, labeled cDNA molecules derived from reference and query RNA samples are co-hybridized to probes arrayed on a glass surface. Gene expression profiles are then calculated for each gene based on the relative hybridization intensities measured between the two samples. The most commonly used reference samples are typically isolates from a single representative RNA source (RNA-0) or pooled mixtures of RNA derived from a plurality of sources (RNA-p). Genomic DNA offers an alternative reference nucleic acid with a number of potential advantages, including stability, reproducibility, and a potentially uniform representation of all genes, as each unique gene should have equal representation in a haploid genome. Using hydrogen peroxide-treated Arabidopsis thaliana plants as a model, we evaluated genomic DNA and RNA-p as reference samples and compared expression levels inferred through the reference relative to unexposed plants with expression levels measured directly using an RNA-0 reference. Our analysis demonstrates that while genomic DNA can serve as a reasonable reference source for microarray assays, a much greater correlation with direct measurements can be achieved using an RNA-based reference sample.     

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX-), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX- were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX- have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real-time RT-PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF-alpha, IL-8 and IL-6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.     

Expression Profile Analysis of Neurodegenerative Disease: Advances in Specificity and Resolution

Microarray technology has become a common tool for developing expression profiles. Initially used in the analysis of cells lines and homogeneous tissues, this platform has been applied to more diverse tissues, such as the brain. Several neural disorders have already been profiled by microarrays using relatively large amounts of tissue. This data has unveiled many genes with differential expression between normal and diseased tissue that could potentially be used as gene markers for these afflictions. Because of the heterogeneity of the CNS, it is likely that small differences between gene expression in these studies would be enhanced by the sampling of a subset of cells based on these newly characterized gene markers. Subtraction of normal, unaffected cells from the sample may also result in a more accurate profile of a diseased cell. Expression profile studies from several neuropathological states are presented, with emphasis placed on those studies using small samples of cellular material and those using specialized methods of cell isolation and RNA amplification.     

Negative feedback buffers effects of regulatory variants

Mechanisms conferring robustness against regulatory variants have been controversial. Previous studies suggested widespread buffering of RNA misexpression on protein levels during translation. We do not find evidence that translational buffering is common. Instead, we find extensive buffering at the level of RNA expression, exerted through negative feedback regulation acting in trans, which reduces the effect of regulatory variants on gene expression. Our approach is based on a novel experimental design in which allelic differential expression in a yeast hybrid strain is compared to allelic differential expression in a pool of its spores. Allelic differential expression in the hybrid is due to cis-regulatory differences only. Instead, in the pool of spores allelic differential expression is not only due to cis-regulatory differences but also due to local trans effects that include negative feedback. We found that buffering through such local trans regulation is widespread, typically compensating for about 15% of cis-regulatory effects on individual genes. Negative feedback is stronger not only for essential genes, indicating its functional relevance, but also for genes with low to middle levels of expression, for which tight regulation matters most. We suggest that negative feedback is one mechanism of Waddington''s canalization, facilitating the accumulation of genetic variants that might give selective advantage in different environments.     

Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells

Numerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like double-stranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.     

Evaluation of gene expression measurements from commercial microarray platforms

Multiple commercial microarrays for measuring genome-wide gene expression levels are currently available, including oligonucleotide and cDNA, single- and two-channel formats. This study reports on the results of gene expression measurements generated from identical RNA preparations that were obtained using three commercially available microarray platforms. RNA was collected from PANC-1 cells grown in serum-rich medium and at 24 h following the removal of serum. Three biological replicates were prepared for each condition, and three experimental replicates were produced for the first biological replicate. RNA was labeled and hybridized to microarrays from three major suppliers according to manufacturers’ protocols, and gene expression measurements were obtained using each platform’s standard software. For each platform, gene targets from a subset of 2009 common genes were compared. Correlations in gene expression levels and comparisons for significant gene expression changes in this subset were calculated, and showed considerable divergence across the different platforms, suggesting the need for establishing industrial manufacturing standards, and further independent and thorough validation of the technology.