External RNA Research Articles

Platelets and platelet-like particles mediate intercellular RNA transfer

The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis.

Abstract 5538: Development of RNA quality control methods to improve BCR-ABL measurement in whole blood samples

Abstract Measurement of BCR-ABL fusion transcripts in whole blood by quantitative, reverse-transcriptase PCR is a clinically validated method to monitor treatment response in patients with chronic myelogenous leukemia. For example, achieving a three-log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A 0.5-log increase from MMR signifies treatment failure sufficient to motivate change in treatment. The purpose of this study was to develop a BCR-ABL test with improved analytical performance characteristics including adequate quality control for RNA degradation or reverse transcription interference and reliable comparison of results across testing sites. To test whether RNA degradation could mask a 0.5-log increase in expression, an RNA degradation model was established by incubating whole blood from each of six individuals with K562 cells at room temperature for various times post-venipuncture. When data from six subjects were combined, the BCR-ABL/103 GUSB value trended down at 24h but was not significantly decreased until 48h (51% decrease, p = 0.004). To quantify the amount of whole blood RNA that can be loaded into an RT reaction without reducing RT efficiency, six different amounts of total RNA extracted from whole blood mixed with K562 cells were added into RT reactions. To measure RT efficiency, an RT Standards Mixture (RTSM) containing known copy numbers of External RNA Control Consortium (ERCC) 171 RNA and ERCC 113 cDNA was added to each RT reaction. RT efficiency was measured as the yield of PCR product from ERCC 171 RNA relative to ERCC 113 cDNA. Based on data from three subjects, compared to RT efficiency at reference concentration of 30 ng/µl RNA in RT, there was a 35% decrease (p = 0.043) at 167 ng/μl RNA and 82% reduction at 1000 ng/μl RNA. The maximum yield of BCR-ABL (molecules/μL cDNA) was observed at 300 ng/µl RNA/RT with a 3-fold increase (p=0.003) compared to 30 ng/µl RNA. Greater than 300 ng/µl RNA/RT did not further increase yield of BCR-ABL molecules/μL cDNA, likely due to reduced RT efficiency. We conclude that BCR-ABL/103 GUSB should be measured within 24 hours following blood collection. Because decreasing RT efficiency is associated with increasing RNA input into RT reactions, including 300 ng/µl RNA in RT reactions provides the maximum sensitivity in measuring BCR-ABL expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5538. doi:1538-7445.AM2012-5538

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles ( Trachemys scripta) using an external RNA control for normalization

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21 °C) and cold-acclimated (5 °C) turtles ( Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression.

Synthetic spike-in standards for RNA-seq experiments

High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2(20) concentration range as spike-in controls to measure sensitivity, accuracy, and biases in RNA-seq experiments as well as to derive standard curves for quantifying the abundance of transcripts. We observed linearity between read density and RNA input over the entire detection range and excellent agreement between replicates, but we observed significantly larger imprecision than expected under pure Poisson sampling errors. We use the control RNAs to directly measure reproducible protocol-dependent biases due to GC content and transcript length as well as stereotypic heterogeneity in coverage across transcripts correlated with position relative to RNA termini and priming sequence bias. These effects lead to biased quantification for short transcripts and individual exons, which is a serious problem for measurements of isoform abundances, but that can partially be corrected using appropriate models of bias. By using the control RNAs, we derive limits for the discovery and detection of rare transcripts in RNA-seq experiments. By using data collected as part of the model organism and human Encyclopedia of DNA Elements projects (ENCODE and modENCODE), we demonstrate that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification. These quality metrics facilitate comparable analysis across different samples, protocols, and platforms.

Assessing affymetrix GeneChip microarray quality

BackgroundMicroarray technology has become a widely used tool in the biological sciences. Over the past decade, the number of users has grown exponentially, and with the number of applications and secondary data analyses rapidly increasing, we expect this rate to continue. Various initiatives such as the External RNA Control Consortium (ERCC) and the MicroArray Quality Control (MAQC) project have explored ways to provide standards for the technology. For microarrays to become generally accepted as a reliable technology, statistical methods for assessing quality will be an indispensable component; however, there remains a lack of consensus in both defining and measuring microarray quality.ResultsWe begin by providing a precise definition of microarray quality and reviewing existing Affymetrix GeneChip quality metrics in light of this definition. We show that the best-performing metrics require multiple arrays to be assessed simultaneously. While such multi-array quality metrics are adequate for bench science, as microarrays begin to be used in clinical settings, single-array quality metrics will be indispensable. To this end, we define a single-array version of one of the best multi-array quality metrics and show that this metric performs as well as the best multi-array metrics. We then use this new quality metric to assess the quality of microarry data available via the Gene Expression Omnibus (GEO) using more than 22,000 Affymetrix HGU133a and HGU133plus2 arrays from 809 studies.ConclusionsWe find that approximately 10 percent of these publicly available arrays are of poor quality. Moreover, the quality of microarray measurements varies greatly from hybridization to hybridization, study to study, and lab to lab, with some experiments producing unusable data. Many of the concepts described here are applicable to other high-throughput technologies.

Abstract 2232: Effectiveness evaluation of an in vitro nucleic acid amplification test for quantification of BCR-ABL fusion transcript variants in human whole blood

Abstract Quantitative measurement of BCR-ABL fusion gene transcripts in whole blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a clinically validated method to monitor tyrosine kinase inhibitor drug response in patients diagnosed with chronic myelogenous leukemia (CML). Monitoring of BCR-ABL expression in blood of cML patients under treatment with a tyrosine kinase inhibitor of BCR-ABL is considered standard of care. A three log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A subsequent 0.5 log increase in BCR-ABL from MMR is considered a sign of treatment failure sufficient to trigger change in treatment. The challenge is to develop a test with sufficient analytical performance characteristics and quality control to reliably measure BCRABL in MMR samples in which there may be less than 10 molecules in 300 of RNA extracted from whole blood. A method validation protocol was developed using the International Committee on Harmonization (ICH) Q2(R1) international validation guidelines. BCRABL variants b2a2 and b3a2 as well as the housekeeping gene GUSB were measured in KCL22 cell line RNA and Stratagene Universal Human Reference RNA test articles in extreme linear dilution assay, robustness testing conditions, and in multiple laboratories. Quality controls were developed to enable loading as much whole blood RNA into the assay as possible without assay interference. These included a reverse transcription (RT) standardization mixture comprising External RNA Control Consortium (ERCC) reagents to control for RT interference, and controls for genomic DNA contamination, RNA integrity, and PCR interference. Results: This test for b2a2 and b3a2 variants of BCR-ABL meets ICHQ2(R1) guidelines for specificity, linearity, accuracy, LOD and LOQ, imprecision, robustness, and reproducibility. Each analyte comprised by the test was linear (R2 > 0.97) over more than 3 logs10, and demonstrated inter-experimental and inter-laboratory imprecision low enough to enable measurement of a 3-fold difference as significant (P <0.05) from a value as low as five molecules/assay. Experiments are underway to quantify the amount of whole blood RNA that can be loaded into RT reaction without interference. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2011-2232

RNA polymerase III transcription in cancer: the BRF2 connection

RNA polymerase (pol) III transcription is responsible for the transcription of small, untranslated RNAs involved in fundamental metabolic processes such mRNA processing (U6 snRNA) and translation (tRNAs). RNA pol III transcription contributes to the regulation of the biosynthetic capacity of a cell and a direct link exists between cancer cell proliferation and deregulation of RNA pol III transcription. Accurate transcription by RNA pol III requires TFIIIB, a known target of regulation by oncogenes and tumor suppressors. There have been significant advances in our understanding of how TFIIIB-mediated transcription is deregulated in a variety of cancers. Recently, BRF2, a component of TFIIIB required for gene external RNA pol III transcription, was identified as an oncogene in squamous cell carcinomas of the lung through integrative genomic analysis. In this review, we focus on recent advances demonstrating how BRF2-TFIIIB mediated transcription is regulated by tumor suppressors and oncogenes. Additionally, we present novel data further confirming the role of BRF2 as an oncogene, extracted from the Oncomine database, a cancer microarray database containing datasets derived from patient samples, providing evidence that BRF2 has the potential to be used as a biomarker for patients at risk for metastasis. This data further supports the idea that BRF2 may serve as a potential therapeutic target in a variety of cancers.

Exploring the use of internal and externalcontrols for assessing microarray technical performance.

BackgroundThe maturing of gene expression microarray technology and interest in the use of microarray-based applications for clinical and diagnostic applications calls for quantitative measures of quality. This manuscript presents a retrospective study characterizing several approaches to assess technical performance of microarray data measured on the Affymetrix GeneChip platform, including whole-array metrics and information from a standard mixture of external spike-in and endogenous internal controls. Spike-in controls were found to carry the same information about technical performance as whole-array metrics and endogenous "housekeeping" genes. These results support the use of spike-in controls as general tools for performance assessment across time, experimenters and array batches, suggesting that they have potential for comparison of microarray data generated across species using different technologies.ResultsA layered PCA modeling methodology that uses data from a number of classes of controls (spike-in hybridization, spike-in polyA+, internal RNA degradation, endogenous or "housekeeping genes") was used for the assessment of microarray data quality. The controls provide information on multiple stages of the experimental protocol (e.g., hybridization, RNA amplification). External spike-in, hybridization and RNA labeling controls provide information related to both assay and hybridization performance whereas internal endogenous controls provide quality information on the biological sample. We find that the variance of the data generated from the external and internal controls carries critical information about technical performance; the PCA dissection of this variance is consistent with whole-array quality assessment based on a number of quality assurance/quality control (QA/QC) metrics.ConclusionsThese results provide support for the use of both external and internal RNA control data to assess the technical quality of microarray experiments. The observed consistency amongst the information carried by internal and external controls and whole-array quality measures offers promise for rationally-designed control standards for routine performance monitoring of multiplexed measurement platforms.

Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements

BackgroundGene expression profiling is an important approach for detecting diagnostic and prognostic biomarkers, and predicting drug safety. The development of a wide range of technologies and platforms for measuring mRNA expression makes the evaluation and standardization of transcriptomic data problematic due to differences in protocols, data processing and analysis methods. Thus, universal RNA standards, such as those developed by the External RNA Controls Consortium (ERCC), are proposed to aid validation of research findings from diverse platforms such as microarrays and RT-qPCR, and play a role in quality control (QC) processes as transcriptomic profiling becomes more commonplace in the clinical setting.ResultsPanels of ERCC RNA standards were constructed in order to test the utility of these reference materials (RMs) for performance characterization of two selected gene expression platforms, and for discrimination of biomarker profiles between groups. The linear range, limits of detection and reproducibility of microarray and RT-qPCR measurements were evaluated using panels of RNA standards. Transcripts of low abundance (≤ 10 copies/ng total RNA) showed more than double the technical variability compared to higher copy number transcripts on both platforms. Microarray profiling of two simulated 'normal' and 'disease' panels, each consisting of eight different RNA standards, yielded robust discrimination between the panels and between standards with varying fold change ratios, showing no systematic effects due to different labelling and hybridization runs. Also, comparison of microarray and RT-qPCR data for fold changes showed agreement for the two platforms.ConclusionsERCC RNA standards provide a generic means of evaluating different aspects of platform performance, and can provide information on the technical variation associated with quantification of biomarkers expressed at different levels of physiological abundance. Distinct panels of standards serve as an ideal quality control tool kit for determining the accuracy of fold change cut-off threshold and the impact of experimentally-derived noise on the discrimination of normal and disease profiles.

Imported Mollusks and Dissemination of Human Enteric Viruses

Imported Mollusks and Dissemination of Human Enteric Viruses

Abstract 825: Development of RNA quality-control methods to improve tissue archiving and molecular diagnostic testing

Abstract Biospecimen collection and processing methods practiced today yield clinical samples with a wide variation in RNA quality. Efforts to improve these methods are inadequate due to insufficient means to measure RNA quality. Improved RNA quality control (QC) measurement methods will enable establishment of cut-off criteria to determine which biospecimens will yield reliable RT-PCR results. Implementation of such criteria will enable existing banks of samples with variable RNA quality to be used for reverse transcriptase polymerase chain reaction (RT-PCR)-based diagnostic test development. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances that either a) interfere with RT efficiency and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. An RNA integrity reference model was established by incubating A549 cell line populations at room temperature for different amounts of time following cytolysis. Between 0 and 100 min. post cytolysis, ACTB degradation, as well as the degradation of other major housekeeping genes, was more rapid than decline in RNA Integrity Number (RIN) Score, a measurement based on microcapillary electropheresis. Quantification of single-copy CC10 gene gDNA in RNA controlled for gDNA contamination and measurement of transcript abundance of each gene relative to known number of respective cDNA internal standards controlled for PCR inhibitors. The RT inhibition test uses a Reverse Transcription Standards Mixture (RTSM) that contains a known number of copies of ERCC (External RNA Control Consortium) RNA and cDNA standards. A known quantity of RTSM is added to an RNA sample and measurement of PCR product from the RNA standard relative to its cDNA standard following RT-PCR assesses RT efficiency. These RT-PCR tests have been implemented on an Agilent 2100 Bioanalyzer® and are being developed for implementation on high throughput RT-PCR platforms such as the Biotrove OpenArray® nanoarray and the Gene Express Standardized Expression Measurement (SEM) Center™. Improved methods for biospecimen collection will be selected and will then be used to identify which samples collected according to present methods are suitable for analysis by RT-PCR-based molecular diagnostic tests. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 825.

Inhibition of U6 snRNA Transcription by PTEN.

PROBLEM STATEMENT: RNA polymerase III (RNA pol III) is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. RNA pol III transcribes both gene internal (tRNA) and gene external (U6 snRNA) promoters and proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB and the RB-related pocket proteins. Also, TFIIIB activity is stimulated by the oncogenes c-Myc and the ERK mitogen-activated protein kinase. Recently, two TFIIIB subunits, BRF1 and BRF2, have been demonstrated to behave as oncogenes, making deregulation of TFIIIB activity and thus RNA pol III transcription an important step in tumor development. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation and survival. Thus, we sought to examine the potential role of PTEN in regulating U6 snRNA transcription. APPROACH: We examined the potential for PTEN to regulate U6 snRNA transcription using in vitro RNA pol III luciferase assays, western blotting and deletion analysis in cancer cell lines differing in their PTEN status. RESULTS: Using breast, cervical, prostate and glioblastoma cancer cells we demonstrate: (1) PTEN inhibition of gene external RNA pol III transcription is cell type specific, (2) PTEN-mediated inhibition of U6 transcription occurs via the C2 lipid-binding domain and (3) PTEN repression of U6 transcription occurs, at least in part, through the TFIIIB subunit BRF2. CONCLUSION/RECOMMENDATIONS: Our data demonstrates that regulation of the U6 snRNA gene by PTEN is mediated, in part by the TFIIIB oncogene BRF2, potentially identifying novel targets for chemotherapeutic drug design.

Inhibition of gene external RNA polymerase III transcription by PTEN

RNA polymerase III is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. Proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB, and the RB‐related pocket proteins. Also, TFIIIB activity is regulated by the oncogenes c‐Myc and the ERK mitogen‐activated protein kinase. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation, and survival. Thus, we examined the potential for PTEN to regulate gene external RNA polymerase III transcription. Here, we report that PTEN inhibits both gene internal and external RNA polymerase III transcription in a variety of cervical, breast and prostate cancer cells. Deletion analysis suggests that the phosphatase domain is important in regulating gene internal RNA polymerase III transcription and the C2‐lipid binding domain in PTEN is important in regulating gene external RNA polymerase III transcription, providing a novel mechanism by which PTEN regulates basal transcription. Additionally, we show that PTEN repression of gene external RNA polymerase III transcription occurs through TFIIIB.

Correlation analysis of external RNA controls reveals its utility for assessment of microarray assay

Quality control of a microarray experiment has become an important issue for both research and regulation. External RNA controls (ERCs), which can be either added to the total RNA level (tERCs) or introduced right before hybridization (cERCs), are designed and recommended by commercial microarray platforms for assessment of performance of a microarray experiment. However, the utility of ERCs has not been fully realized mainly due to the lack of sufficient data resources. The US Food and Drug Administration (FDA)-led community-wide Microarray Quality Control (MAQC) study generates a large amount of microarray data with implementation of ERCs across several commercial microarray platforms. The utility of ERCs in quality control by assessing the ERCs’ concentration–response behavior was investigated in the MAQC study. In this work, an ERC-based correlation analysis was conducted to assess the quality of a microarray experiment. We found that the pairwise correlations of tERCs are sample independent, indicating that the array data obtained from different biological samples can be treated as technical replicates in analysis of tERCs. Consequently, the commonly used quality control method of applying correlation analysis on technical replicates can be adopted for assessing array performance based on different biological samples using tERCs. The proposed approach is sensitive to identifying outlying assays and is not dependent on the choice of normalization method.

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

BackgroundReference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.MethodsSix genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software.ResultsHMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.ConclusionOf six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.

Expression of genes involved in excitatory neurotransmission in anoxic crucian carp (Carassius carassius) brain

The crucian carp, Carassius carassius, survives months without oxygen. During anoxia it needs to keep energy expenditure low, particularly in the brain, with its high rate of ATP use related to neuronal activity. This could be accomplished by reducing neuronal excitability through altered expression of genes involved in excitatory neurotransmission. Through cloning and the use of a recently developed real-time RT-PCR approach, with an external RNA control for normalization, we investigated the effect of 1 and 7 days of anoxia (12 degrees C) on the expression of 29 genes, including 8 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, 6 N-methyl-d-aspartate (NMDA) receptor subunits, 7 voltage-gated sodium and calcium channels, 4 glutamate transporters, and 4 genes involved in NMDA receptor-mediated neuroplasticity. The subunits of the majority of the gene families had expression profiles similar to those observed in the mammalian brain and showed remarkably stable expression during anoxia. This suggests that the genes may have similar functions in crucian carp and mammals, and that the excitatory abilities of the crucian carp brain are retained during anoxia. Although the data generally argue against profound neural depression ("channel arrest"), NMDA receptor subunit (NR) expression showed features that could mediate reduced neural excitability. Primarily, the NR2 subunit expression, which was dominated by NR2B and NR2D, resembled that seen in hypoxia-tolerant neonatal rats, and decreased anoxic expression of NR1, NR2C, and NR3A indicated reduced numbers of functional NMDA receptors. We also report the full-length sequence of crucian carp NR1 mRNA and a novel NR1 splice cassette introducing an N-glycosylation site into the extracellular S1S2 domain.

Improved normalization of real-time reverse transcriptase polymerase chain reaction data using an external RNA control

No golden standard exists for normalization of real-time reverse transcriptase polymerase chain reaction (RT PCR) data and procedures used are often not validated. Numerous studies have indicated that current approaches are inadequate. Here, we report the development of an external RNA control approach. It is the first to add external RNA to tissue on a per unit weight basis, and we demonstrate its accuracy, suitability, and necessity in experiments involving severe physiological challenges. We utilized the approach to examine the expression of the internal RNA control genes (reference genes) β-actin, cyclophilin A, and glyceraldehyde 3-phospate dehydrogenase in brain and heart of normoxic and anoxic crucian carp (Carassius carassius). The internal RNA control genes differed significantly in expression in experimental groups, especially in heart. We also demonstrate that the external RNA control approach provides a more accurate normalization of target genes. For example, it revealed a 2.5-fold increase in the expression of the stress-response gene HSC70, which was not detected using β-actin or geNorm. Further, we demonstrate and discuss the need for using the optimized and standardized external RNA control protocol reported. Collectively, our data suggest that the normalization of real-time RT PCR data is considerably improved by adding an external RNA control to the samples.

Differential expression of the TFIIIB subunits Brf1 and Brf2 in cancer cells

BackgroundRNA polymerase (pol) III transcription is specifically elevated in a variety of cancers and is a target of regulation by a variety of tumor suppressors and oncogenes. Accurate initiation by RNA pol III is dependent on TFIIIB. In higher eukaryotes, two forms of TFIIIB have been characterized. TFIIIB required for proper initiation from gene internal RNA pol III promoters is comprised of TBP, Bdp1, and Brf1. Proper initiation from gene external RNA pol III promoters requires TBP, Bdp1, and Brf2. We hypothesized that deregulation of RNA polymerase III transcription in cancer may be a consequence of altered TFIIIB expressionResultsHere, we report: (1) the TFIIIB subunits Brf1 and Brf2 are differentially expressed in a variety of cancer cell lines: (2) the Brf1 and Brf2 promoters differ in activity in cancer cell lines, and (3) VAI transcription is universally elevated, as compared to U6, in breast, prostate and cervical cancer cells.ConclusionDeregulation of TFIIIB-mediated transcription may be an important step in tumor development. We demonstrate that Brf1 and Brf2 mRNA are differentially expressed in a variety of cancer cells and that the Brf2 promoter is more active than the Brf1 promoter in all cell lines tested. We also demonstrate, that Brf1-dependent VAI transcription was significantly higher than the Brf2-dependent U6 snRNA transcription in all cancer cell lines tested. The data presented suggest that Brf2 protein expression levels correlate with U6 promoter activity in the breast, cervical and prostate cell lines tested. Interestingly, the Brf1 protein levels did not vary considerably in HeLa, MCF-7 and DU-145 cells, yet Brf1 mRNA expression varied considerably in breast, prostate and cervical cancer cell lines tested. Thus, Brf1 promoter activity and Brf1 protein expression levels did not correlate well with Brf1-dependent transcription levels. Taken together, we reason that deregulation of Brf1 and Brf2 expression could be a key mechanism responsible for the observed deregulation of RNA pol III transcription in cancer cells.

Detailed Technical Analysis of Urine RNA-Based Tumor Diagnostics Reveals ETS2/Urokinase Plasminogen Activator to Be a Novel Marker for Bladder Cancer

The noninvasive detection of RNA tumor markers in body fluids represents an attractive diagnostic option, but diagnostic performance of tissue-derived markers is often poorer when measured in body fluids rather than in tumors. We aimed to develop a procedure for measurement of tumor RNA in urine that would minimize donor-dependent influences on the results. RNA isolated from urinary cell pellet, cell-depleted fraction, and whole urine was quantified by reverse transcription quantitative-PCR. The donor-dependent influence of urine background on individual steps of the standardized procedure was analyzed using an external RNA standard. Using a test set of samples from 61 patients with bladder cancer and 37 healthy donors, we compared 4 putative RNA tumor markers identified in whole urine with 5 established, tissue-derived RNA tumor markers for the detection of bladder cancer. Of the markers analyzed by this system, the RNA ratio of v-ets erythroblastosis virus E26 oncogene homolog 2 (avian; ETS2) to urokinase plasminogen activator (uPA) enabled the most specific (100%) and sensitive (75.4%) detection of bladder cancer from whole urine, with an area under the curve of 0.929 (95% CI 0.882-0.976). The described methodology for RNA marker detection in urine appears to be clinically applicable. The ratio of ETS2 mRNA to uPA mRNA in urine is a potential marker for bladder cancer.

Folding Cooperativity in RNA and DNA Is Dependent on Position in the Helix

Secondary structural motifs play essential roles in the folding and function of RNA and DNA molecules. Previous work from our lab compared the folding of small DNA and RNA hairpin loops containing a sheared GA pair [Moody, E. M., Feerar, J. C., and Bevilacqua, P. C. (2004) Biochemistry 43, 7992-7998]. We found that the small DNA hairpins fold in a highly cooperative manner with indirect coupling, while their RNA counterparts fold in a much less cooperative fashion and display direct coupling. Herein, we extend this study to the double-stranded helix. We carried out double mutant cycles on base pairs having identical nearest-neighbor contexts but located in either external or internal helical registers. In the external register, both RNA and DNA exhibit extensive folding cooperativity between the penultimate and terminal base pair, which is independent of mismatch identity. In contrast, DNA exhibits virtually no folding cooperativity in the center of the helix, while RNA maintains substantial coupling, which is dependent on mismatch identity. Two models account for these non-nearest-neighbor effects: one involves the unfavorable entropy of helix initiation common to DNA and RNA, and the other involves steric and electrostatic strain peculiar to RNA. These data show that RNA can display cooperativity less than, greater than, or equal to that of DNA depending on context and position.