Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes.

In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.

Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay.

OBJECTIVES: To detect Cochlodinum polykrikoides in long-term monitoring and high-throughput sampling projects using an integrated sandwich hybridization and nuclease protection assay (NPA-SH). RESULTS: The specificity of the probes was verified with individual and mixed cultures as well as field collection, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. CONCLUSIONS: This approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1ß, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1ß, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Explanatory chapter: nuclease protection assays.

Nuclease protection assays are a highly sensitive, solution-based technique used to detect and quantify specific RNA targets from complex RNA mixtures. Today, this technique is frequently performed using kits, and the following chapter will explain the principles of how these kits work and some considerations to keep in mind when using them.

Ribonuclease protection assay on microchip electrophoresis.

We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.

A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression.

The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research.

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.

Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay.

Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. Studies have shown that NATs may participate in a broad range of gene regulatory events. The identification of NATs in human, mouse and Escherichia coli has revealed their widespread occurrence in both eukaryotic and prokaryotic life. However, little is known about NATs in Helicobacter pylori (H. pylori), a human pathogen which is associated with gastric diseases. Here we systematically screened NATs in H. pylori by a novel experimental strategy based on RNase I protection assay. We successfully constructed a cDNA library of NATs and developed a novel poly(A)-tailed RT-PCR method to monitor the expression of NATs. After sequencing, bioinformatic analysis and expression detection, two novel NATs (NAT-39 and NAT-67) were confirmed. They were, respectively, complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). Taken together, the results suggest that NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. Pylori in a sequence complementary manner with target mRNAs.

Ribonucleases.

Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed.

RNA polymerases.

This unit describes DNA-dependent, RNA-dependent, and template-independent RNA polymerases. DNA-dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA-dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template-independent polymerase, catalyzes the incorporation of AMP residues onto the free 3'-hydroxyl terminus of RNA, utilizing ATP as a precursor. Specific reaction conditions of poly(A) polymerase, as well as applications including RNA tailing and 3' end labeling, are discussed.

Effects of chronic central leptin infusion on proopiomelanocortin and neurotensin gene expression in the rat hypothalamus.

Leptin signaling in the hypothalamus is critical for normal food intake and body weight regulation. While hyperleptinemia in obese people suggests a state of leptin resistance, the mechanism is not clearly understood. In a rat model of central leptin infusion in which animals develop resistance to the satiety action of leptin, orexigenic peptide producing neuropeptide Y neurons in the hypothalamus develop leptin resistance. However, it is still unknown if increased hypothalamic leptin tone caused by central leptin infusion results in the development of leptin resistance in anorexigenic peptide producing proopiomelanocortin (POMC) and neurotensin (NT) neurons. To this end, male rats were infused chronically with leptin (160 ng/h) or vehicle into the lateral cerebroventricle for 16 days. On day 4 of leptin infusion when food intake was decreased, POMC and NT mRNA levels, as determined by RNAse protection assay, were significantly increased as compared to control. By contrast, on day 16 of leptin infusion, when food intake was mostly normalized, both POMC and NT mRNA levels remained unchanged compared with control. These findings suggest the development of leptin resistance in the POMC and NT neurons following chronic elevation of hypothalamic leptin tone, which may be involved in the development of resistance to the satiety action of leptin following central infusion of this peptide hormone.

Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH).

Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China. In this study, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was used to qualitatively and quantitatively detect P. globosa. Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields. The linear regression equation for P. globosa was obtained, and the lowest detection number of cells was 1.8 x 10(4) cells. Statistics showed that there was no distinct difference between the results of detecting the microalgae by NPA-SH and traditional microscopy. This technique has good reliability, accuracy, and can give a remarkably high sample processing rate. Sandwich hybridization integrated with nuclease protection assay will provide an efficient alternative to microscopic method for monitoring and investigating the bloom of P. globosa.

[A novel strategy for systematic identification of natural antisense transcripts of Pseudomonas aeruginosa based on RNase I protection assay].

Natural antisense transcripts (NATs) are widespread in prokaryotes and eukaryotes. They have very important functions in regulating expression of their target genes. However, until now, NATs haven't been systematic identified in high throughput for a lack of effective experimental method. Here, we have developed a strategy based on RNase I protection assay, which permit us to identify NATs in large scale which presented inside Pseudomonas aeruginosa. After being isolated from total RNA of P. aeruginosa strain ATCC 6872 (PAO1), NATs that encoded chromosomally were cloned in a simple and efficient way and a NATs gene library was constructed successfully. 78 random ly selected positive clones were sequenced and analyzed by bioinformatic tools. There are several candidate molecules which are located in PAO1 genome precisely. This study suggests that there may be exist some gene regulatory mechanism acted by NATs in PAO1 and this worth further analysis.

Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma.

Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to approximately 1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R(2)>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R(2)=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

RNase protection assay for quantifying gene expression levels.

Quantifying the level of mRNA is central to the study of mammalian gene expression. Conventional approaches such as Northern blotting are often prone to low sensitivity and reproducibility. The RNase protection assay (RPA) provides a sensitive alternative for the detection and quantification of mRNA. The RPA is based on the hybridization in solution of a labeled single-stranded antisense RNA probe with a target mRNA. After hybridization, single-strand specific RNases are then used to digest away unhybridized RNA. The hybrid can be resolved by a denaturing gel. Subsequent detection will reveal the appropriate-sized gel band corresponding to the target mRNA. The major advantage of RPA is the high sensitivity and the simultaneous detection and quantification of multiple mRNA targets in a single RNA sample. The primary limitation of RPA is the lack of information on transcript size.

Mapping transcriptional start sites and in silico DNA footprinting.

Determination of a gene's transcriptional start site underlies the identification of the proximal promoter region and thus facilitates the subsequent analysis of components controlling its expression, namely, cis-acting regulatory elements and their cognate binding proteins. It also enables assembly of meaningful reporter constructs to examine promoter function in different cellular contexts. In this chapter, basic protocols for two experimental approaches to transcriptional start site determination are described: primer extension analysis and the ribonuclease protection assay. Consideration is also given to RNA sources, RNA purification, and primer design. The explosion in genomic DNA and mRNA sequence information derived from genomic sequencing projects, expressed sequence tags and microarrays, combined with in silico analysis, such as automated sequence annotation and gene identification algorithms, now provides an alternative source of detailed information on gene structure and expression. Two approaches to the in silico identification of transcription factor binding sites are described.

Structural and biochemical basis for misfolded RNA recognition by the Ro autoantigen.

The Ro autoantigen is ring-shaped, binds misfolded noncoding RNAs and is proposed to function in quality control. Here we determine how Ro interacts with misfolded RNAs. Binding of Ro to misfolded precursor (pre)-5S ribosomal RNA requires a single-stranded 3' end and helical elements. As mutating most sequences of the helices and tail results in modest decreases in binding, Ro may be able to associate with a range of RNAs. Ro binds several other RNAs that contain single-stranded tails. A crystal structure of Ro bound to a misfolded pre-5S rRNA fragment reveals that the tail inserts into the cavity, while a helix binds on the surface. Most contacts of Ro with the helix are to the backbone. Mutagenesis reveals that Ro has an extensive RNA-binding surface. We propose that Ro uses this surface to scavenge RNAs that fail to bind their specific RNA-binding proteins.

Determinação do perfil de expressão dos mRNAs da família das Smads e dos componentes do complexo AP-1 em carcinoma de célula escamosa de cabeça e pescoço / Smads and AP-1 messenger RNA expression pattern in head and neck squamous cell carcinoma

A expressão de Smads e de membros da família AP1/ jun-fos podem refletir alterações da via de TGF, uma via importante para o câncer epidermóide de cabeça e pescoço (HNSCC). Encontramos expressão aumentada dos mRNAs das Smads1-8 em HNSCC em comparação com tecido normal adjacente, por RPA. Além disso, as curvas de sobrevida de Kaplan Meier e a análise multivariada mostraram que a Smad6+ parece ser um fator determinante de bom prognóstico em HNSCC. Quanto a família AP-1, mensurado por Northern blot, somente Fra-1 mostrou-se aumentado no tumor e associado à presença de linfonodos comprometidos. Nossos dados sugerem que a positividade de Smad6 possa ser marcador de bom prognóstico em HNSCC / Smad and AP1 messenger RNA expression may underlie disruptions affecting TGF signaling in head and neck squamous cell carcinoma (HNSCC). Analysis of Smads1-8 mRNA expression by RPA has shown Smad expression is globally increased in tumor as compared to adjacent normal tissue. Kaplan Meier survival curves and multivariate analysis revealed that Smad6 positivity in tumor was an independent good prognostic factor in HNSCC. In relation to AP-1, as measured by Northern blot, only Fra-1 was overexpressed in tumor and directly related to the presence of lymph node involvement. Our data suggest that Smad6 may be a marker of good prognosis in HNSCC...

Characterization of a mammalian smooth muscle cell line that has retained transcriptional and posttranscriptional potencies.

Unlike skeletal and cardiac muscle cells that differentiate irreversibly, smooth muscle cells (SMCs) retain a high degree of plasticity. During the so-called phenotypic modulation, SMCs can undergo transition between a contractile phenotype and a highly proliferative synthetic phenotype, as apparent from the extinction of numerous smooth muscle (SM) markers when they are passaged in culture. It would be very useful to have an SMC line that can be indefinitely propagated for the cellular and molecular analysis of the mechanisms that underlie the control of SM differentiation. This report describes an immortalized rabbit aorta SMC-derived cell line (U8A4) that has conserved differentiated properties through multiple subcultures. U8A4 cells can grow in the absence of serum and express the SMC markers studied, including SM alpha-actin, SM calponin, SM22alpha, SM alpha-tropomyosin (alpha-TM), SM myosin heavy chain (SM-MHC), and myocardin. U8A4 cells can activate SMC-restricted promoters like those of SM22alpha, SM calponin, and SM-MHC genes as efficiently as described previously for rat SMC lines (PAC1, A7r5, and A10). These cells can also process exogenous alpha-TM transcripts according to an SM-specific pattern. These results demonstrate that the U8A4 cell line constitutes a good alternative model to existing SMC lines that could facilitate the study of the transcriptional and posttranscriptional regulatory mechanisms underlying SMC differentiation.

Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay.

Currently, methods for protein detection are not as sensitive and specific as methods for detection of specific nucleic acid sequences. Here, we present an analogous technique for detection of proteins using aptamers as ligands for target binding. We have named this method the aptamer-based exonuclease protection assay. We applied a special oligonucleotide probe containing a thrombin aptamer, which has the capacity to recognize thrombin with high affinity and specificity. The aptamer probe is a 22-base-long single-strand oligonucleotide with the thrombin aptamer sequence at the 3'-terminus and 7 additional nucleotides at the 5'-terminus, which is able to bind thrombin with high affinity and specificity. In the exonuclease protection assay, thrombin binds the aptamer and thereby protects it from degradation by exonuclease I, whereas any unbound aptamer probe is degraded by exonuclease I. Subsequently, the aptamer probes that were protected from exonuclease I by thrombin act as linkers to join two free connectors, which contain sequences matching the probe. The joined products, which reflect the identity and amount of the target protein, are amplified by PCR. The exonuclease protection assay is extremely sensitive, since it is based on PCR amplification. This method can detect as few as several hundred molecules of target protein without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.