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DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor ® 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye–dye or dye–DNA–dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients ( R 2 ) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.

The primary goal of the Phase II Contract was to continue to design, manufacture, and test real time polymerase chain reaction (qPCR) reagents for detection of one or more of six potentially deadly arbovirus pathogens: Dengue, Rift Valley Fever, Chikungunya, Crimean-Congo Hemorrhagic Fever, Sand Fly Fever, and Tick-Borne Encephalitis viruses. The qPCR reagents being provided in Phase II are additional primers and probes ( signature sets ) sets for qPCR applications, as well as positive controls (synthetic targets) for the assays. Work product in Phase II included: (1) design and synthesis of a synthetic target representing all four Dengue serotypes; (2) cloning of a plasmid control for the new RT-qPCR signature set; (4) qPCR testing of the new Dengue signature set, synthetic template, and plasmid control; (5) determination of efficiency, Linearity of Range (LOR), and Limit of Detection (LOD) for all eight qPCR signature sets; (6) bioinformatic analysis and alignment of sequences published for the high priority agents listed; (7) design and synthesis of a new consensus qPCR signature set for Dengue types 1-4 ( DENcon ) based on bioinformatics analysis of 42 Dengue subtypes; (8) design and synthesis of a synthetic template for the consensus Dengue signature set; (9) design and synthesis of a control plasmid ( ARBO9 ) containing sites for all eight signature sets; (10) the testing of RNA generated from ARBO9 as a synthetic target; and (11) stability testing of all reagents, including positive controls.

In situ hybridization histochemistry (ISHH) using synthetic oligodeoxynucleotide probes has been used to demonstrate the sites of expression of mRNA for vasopressin (AVP) and oxytocin (OXT) in the rat brain. ISHH was performed with two types of non-radioactive probes, labelled with either alkaline phosphatase or 5′-biotin. Simultaneous detection of the mRNAs for both AVP and OXT was achieved using an alkaline phosphatase substrate for the AVP probe and an anti-biotin monoclonal (mouse) antibody for the OXT probe. These probes revealed two non-overlapping populations of AVP and OXT neurons on the same section.

Stellaris ™ fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection

The genome is extensively transcribed, yet more than 50 % of RNA transcripts lack protein coding potential. In recent years, long noncoding RNAs (lncRNAs) have been shown to play extensive roles in all the central aspects of gene function including imprinting, epigenetic regulation, transcription, splicing and nuclear/cytoplasmic trafficking, translation and RNA stability, with wide ranging effects on cell cycle and differentiation. Due to the increasingly central role of lncRNAs in gene transcription and disease states, it is important to develop tools to allow direct visualization and quantification of lncRNAs that could be useful both in the basic science arena and in the clinical setting with possible uses both in diagnostics and prognostics. We recently streamlined both the design and synthesis of fluorescently labeled probe sets containing multiple tiled 20-mer GC-balanced oligonucleotides for use in RNA fluorescence in situ hybridization (RNA FISH). In contrast to traditional cell-disruptive RNA analyses (e.g. northern blotting and qPCR) that report relative population averages of RNA content, single molecule RNA FISH affords specific and sensitive detection of RNAs and quantitatively establishes the intracellular localization of the target lncRNAs. We have validated an imaging based platform to perform RNA FISH in both manual (coverslip) and automated (384 well) formats that allows tremendous multiplexing and throughput using high throughput microscopy. Moreover, we developed high content analysis routines that permit quantification of RNA particles at the single cell level and determine their localization and pattern (texture). As specific lncRNAs (e.g. MALAT1) are known to target specific intranuclear domains (e.g. splicing speckles) we developed classification methods based upon sets of intensity and texture features to compare intracellular patterns of novel/less characterized ncRNAs. Moreover, this framework will also permit to address changes in lncRNA expression and patterns during the cell cycle since large number of cells can be analyzed simultaneously. We are now expanding our analysis to primary tumor tissues where lncRNAs patterns will be correlated with known FISH-compatible biomarkers, thus providing a picture simultaneously for intra- and inter-cell variation. In summary, the results and tools presented here will enable the study of disease related lncRNAs in a high content, high throughput mode. These efforts will enable gaining better understanding of how expression and localization of lncRNAs may contribute to the diseased phenotype, and add a large selection of novel biomarkers that can be interrogated for improved diagnosis, prognosis, and therapeutic choice. Citation Format: Hans E. Johansson, Arturo V. Orjalo, Sally R. Coassin, Jerry. L. Ruth, Fabio Stossi, Michael A. Mancini. High-throughput imaging and high content analysis of disease relevant lncRNAs examined by RNA fluorescence in situ hybridization (RNA FISH). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2013-1940

The simple use of nonisotopic hybridization probes to detect complementary sequences provides valuable information in a large number of research and commercial applications. In hybridization assays, the four "S's (speed, simplicity, sensitivity, and specificity) are important criteria for determining the choice of probe and label. The direct chemical combination of synthetic oligonucleotide probes and enzyme labels offer advantages unmatched by other approaches, with the oligonucleotide providing rapid hybridization and high specificity, and the direct enzyme label providing simple and sensitive detection. Such oligonucleotide-enzyme conjugates ("oligozymes") can be used in a variety of hybridization and detection formats, including dot blots, Southern/northern blots, in situ, and solution hybridization/capture schemes. The practical synthesis and use of such oligozymes are summarized.

[2-14C]5-Allyl-2′-deoxyuridine was synthesized directly from [2-14C]2′-deoxyuridine using mercury, palladium, and 3-chloropropene. [2-14C]5-Propyl-2′-deoxyuridine was obtained by hydrogenation of the [14C]5-allyl-2′-deoxy-uridine. Advantages of the synthetic method and its application to the preparation of other radiolabeled 5-alkyl/alkeny1-2′-deoxyuridines are discussed.

In situ hybridization histochemistry has been used successfully by many laboratories to detect mRNAs within single cells in the CNS. The detection of these hybrids in CNS tissue sections has been accomplished mainly with radioactive probes. However, radiolabeled probes have intrinsic limitations, including the long exposure time required for high resolution autoradiography and the inability to detect multiple RNA species within the same neuron. Here we report a new method to detect mRNA in situ using a synthetic DNA probe conjugated to alkaline phosphatase (AP). The probe was synthesized to be complementary to the glycoprotein coding region of vasopressin mRNA. Under normal hybridization conditions high resolution detection of vasopressin mRNA within individual neurons was routinely obtained within 8 h. The distribution of hybridization signal obtained with the AP-conjugated probe was identical to that observed with the same probe radiolabeled with [35S]dATP. Hybridization-positive neurons were found in all regions of the CNS that have been previously reported to synthesize vasopressin, including magnocellular neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus. Small diameter neurons were also observed in the suprachiasmatic nucleus, the accessory PVN, the bed nucleus of the stria terminalis, and a cell group along the ventral surface of the optic tract. These results suggest that nonradioactive detection of neuropeptide mRNA in situ can be easily accomplished within 24 h. Furthermore, the improved resolution with AP-conjugated oligonucleotide probes should enhance efforts to study the regulation of gene expression in the nervous system.

The metabolism and disposition of 5-propyl-2'-deoxyuridine (Pr-dUrd) in herpes simplex virus type 1 infections were investigated in cell culture using [14C]Pr-dUrd, [32P]orthophosphate, and several methods including high pressure liquid chromatography and isopycnic centrifugation. Results in infected cells indicate Pr-dUrd 1) is taken up and phosphorylated to mono-, di-, and triphosphates; 2) is incorporated into DNA; 3) preferentially inhibits synthesis of viral DNA; 4) blocks re-initiation of viral DNA synthesis even after removal of the nucleoside from the culture; and 5) exerts these effects early in the course of infection (before 6 h postinfection). Pr-dUrd was not phosphorylated in uninfected cells, and had little or no effect on apparent cellular DNA synthesis in infected or uninfected cells. Present evidence suggests one possible antiviral event could be the lethal effect of Pr-dUrd after incorporation into viral DNA by alteration of DNA template-directed functions such as replication.

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.

Effects of the triphosphates of eight pyrimidine nucleoside analogs (5-substituted, 2'-fluoroara-, and acyclonucleosides) and acycloguanosine were examined on the ribonucleotide reductases prepared from uninfected and herpes simplex virus (types 1 and 2)-infected HeLa cells. Of the analogs tested, E-5-propenyl- and E-5-(2-bromovinyl)-dUTP were more potent inhibitors than dTTP of the enzymes from virus-infected cells, whereas only the former compound showed this effect on the uninfected HeLa enzyme.

E-5-Propenyl-2'-deoxyuridine (E-5-propenyl-dUrd) inhibited the growth of herpes simplex virus (HSV) types 1 (HSV-1) and 2 in culture. The concentration of drug required to give a 2-log reduction in virus titer was 5 microM for HSV-1 and 23 microM for HSV-2. The anti-HSV-1 activity of this agent was more potent than 5-propyl-dUrd, equivalent to E-5(3,3,3-trifluoropropenyl)-dUrd, and less potent than E-5-bromovinyl-dUrd. The HSV-1 mutant (B2006) lacking the ability to induce virus-specific thymidine kinase could not be inhibited by E-5-propenyl-dUrd. The binding constants of E-5-propenyl-dUrd to HSV-1, HSV-2, varicella-zoster virus, and human mitochondrial thymidine kinases were established to be 0.2, 6.2, 0.3, and 0.8 microM, respectively. Thymidine phosphorylation catalyzed by human cytosol thymidine kinase could not be inhibited by E-5-propenyl-dUrd at a concentration 10-fold higher than the thymidine in the assay. When thymidine and E-5-propenyl-dUrd were added concomitantly at equal concentrations to virus-infected cells, the antiviral activity was not reversed in HSV-1 and only partially reversed in HSV-2. E-5-Propenyl-dUrd also inhibited the growth of human cells in culture with 50% inhibitory dose of 50 microM. Since this inhibition could be readily reversed by a lower concentration of thymidine, the idea of selective protection is proposed. This approach could avoid the cytotoxic effect of an antiviral agent with properties similar to E-5-propenyl-dUrd without sacrificing antiviral activity.

Three single-stranded oligonucleotide probes, 22 bases long, homologous to unique regions of herpes simplex virus (HSV) types 1 (HSV-1) and 2 (HSV-2) and a region common to both were chemically synthesized with use of a modified phosphochloridite protocol. For hybridization experiments each probe was labeled with use of polynucleotide kinase and [gamma-32P] ATP to a specific activity of approximately 2 X 10(9) cpm/micrograms. Two hundred one clinical isolates of HSV (96 HSV-1 and 105 HSV-2) collected from vesicles in the mucocutaneous junction of the mouth or from the genital area were analyzed. There was a 99% (199 of 201) agreement between hybridization and monoclonal antibody typing; the two discrepant isolates of HSV-2 that were negative by monoclonal antibody typing were confirmed as HSV-2 by restriction endonuclease analysis. The probes detected between 10(4) and 10(5) HSV infectious units and from 150 to 600 HSV-infected Vero cells. No binding was detected between any of the three probes and isolates of cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus.