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Smailus DE, Marziali A, Dextras P, Marra MA, Holt RA. Simple, robust methods for high-throughput nanoliter-scale DNA sequencing. Genome Research 15;2005:1447–1450. [PubMed]
Large-scale DNA sequencing projects, especially genomic sequencing and polymorphism detection sequencing, remain very costly enterprises. In conventional Sanger sequencing with four-color fluorescence and capillary electrophoresis, reaction volumes of several microliters are typically used, yet such reactions yield quantities of products far in excess of requirements. The limitations on volume reduction include microtiter plates that allow sample loss through seal leakage and condensation on unwetted inner surfaces of the wells, and the limited capabilities of robotic devices that are used to dispense liquid to the conventional microtiter plate formats. This paper describes robust, high-throughput procedures by which reaction volumes are reduced to 400 nL. They include the use of SPRI Plug Low Volume Lids (Agencourt Bioscience) for sealing plates to reduce residual airspace in wells and thus reduce sample loss by evaporation. Liquid volumes in the nanoliter range are delivered to the wells using an Aurora Discoveries Flying Reagent Dispenser. This instrument was developed for high-throughput screening in the pharmaceutical industry, but has not previously been applied to DNA sequencing. Read lengths (PHRED20 bases) of 765 ± 172 for plasmid clones, 621 ± 201 for fosmid clones, and 647 ± 189 for BAC clones are reported. The changes provide an approximately 10-fold reduction in reagent use compared with standard protocols.
Ehrich M, Nelson MR, Stanssens P, Zabeau M, Liloglou T, Xinarianos G, Cantor CR, Field JK, van den Boom D. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proceedings of the National Academy of Sciences, U.S.A. 102;2005:15,785–15,790.
A methodology for analysis of DNA methylation is proposed that involves base-specific cleavage of single-stranded DNA and MALDI-TOF mass spectral analysis. Genomic DNA is treated with bisulfite to convert unmethylated cytosine to uracil. DNA regions containing CpG islands that represent potential sites of DNA methylation are then amplified by PCR using primers located outside the target sequence. One PCR primer is tagged with a T7 promoter sequence to allow subsequent transcription of the PCR product into RNA. The transcript is cleaved in a base-specific manner by an endoribonuclease. The conversion of unmethylated cytosine to uracil during bisulfite treatment results in the production of cleavage products that reflect the underlying methylation pattern. The cleavage products are analyzed by MALDI-TOF mass spectrometry. Preferential amplification of methylated or unmethylated DNA is avoided by excluding CpG sites from the primer region. A combination of cleavage reactions enables complete evaluation of DNA methylation, including discovery of methylated genes, analysis of methylation patterns, and relative quantitation of methylated and unmethylated sequences. The method is suitable for high-throughput, automated methylation analysis.
The international HapMap Consortium. A haplotype map of the human genome. Nature 437;2005:1299–1320. [PubMed]
The International HapMap Project is creating a resource to accelerate the identification of genetic factors that influence medical traits. The database of common variation in the human genome described in this paper is the result of the first phase of the project. Genotypes for 269 individuals from four populations are ascertained with respect to more than one million single nucleotide polymorphisms (SNPs). The paper documents the generality of recombination hotspots and long segments of strong linkage disequilibrium, and shows that a low level of haplotype diversity exists among individuals. The data will guide genetic association studies, and make possible the identification of deletion variants in the human genome. They will assist in the investigation of fine-scale recombination and help identify regions that have been subject to natural selection.
Xia B, Kawar ZS, Ju T, Alvarez RA, Sachdev GP, Cummings RD. Versatile fluorescent derivatization of glycans for glycomic analysis. Nature Methods 2;2005:8445–8850.
The construction of glycan arrays for investigating carbohydrate function has been impaired by a lack of facile chemistry to activate small quantities of free reducing glycans to form derivatives with primary amines that can be used for conjugation. In this paper, a simple method is described in which glycans are derivatized with 2,6-diaminopyridine (DAP) to generate glycans that contain a primary amine for further conjugation. The chemistry also provides fluorescent derivatives for quantitation and visualization on microarray surfaces. A wide variety of glycans are shown to be amenable to derivatization in this way, and to bind appropriate carbohydrate-binding proteins (CBPs). This technique is expected to expand the use of glycan arrays and the diversity of glycans available for investigating carbohydrate-binding protein specificity and pathogen recognition.
Parker LL, Schilling AB, Kron SJ, Kent SBH. Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection. Journal of Proteome Research 4;2005:1863–1866. [PubMed]
In studies of the phosphoproteome in vivo and in vitro, an advantageous technique is to get a kinase of interest to add thiophosphate instead of phosphate, and makes use of the phosphatase resistance of the resulting analog to cause build-up of downstream signaling intermediates, or exploits the chemically reactive thiophosphate to add fluorescent probes for phosphorothioate target detection. Unfortunately, many kinases show poor kinetics with the required substrate, ATPγS, making it difficult to form thiophosphates in good yield, especially when the preferred substrate, ATP, is present along with Mg2+ as the normal cofactor. This paper presents a process that employs MALDI-TOF mass spectrometry as an assay technique by which efficient thiophosphorylation conditions can be identified for any kinase of interest in the presence of ATP and Mg2+. Thiophosphorylation by Abl kinase is shown to be rescued by adding small amounts of Mn2+, while protein kinase A responds best to Co2+.
Wang HJ, Jackson SN, McEuen J, Woods AS. Localization and analysis of small drug molecules in rat brain tissue sections. Analytical Chemistry 77;2005:6682–6686. [PubMed]
MALDI-MS is shown to provide a simple approach to determining the in situ localization of drug molecules in tissue sections. Following intracranial injection of chlorisondamine (a nicotine receptor blocker) or intraperitoneal injection of cocaine, the brains of rats are snap-frozen and then sectioned in a cryostat. The sections are melted onto a target plate, and a suitably chosen MALDI matrix is spotted onto them for mass spectral analysis. The identity of the drugs is confirmed by MS/MS. This approach provides the potential for imaging, pharmacokinetics, and toxicology applications.
Touboul D, Kollmer F, Niehuis E, Brunelle A, Laprévote O. Improvement of biological time-of-flight secondary ion mass spectrometry imaging with bismuth cluster ion source. Journal of the American Society for Mass Spectrometry 16;2005:1608–1618. [PubMed]
Ostrowski SG, Szakal C, Kozole J, Roddy TP, Xu J, Ewing AG, Winograd N. Secondary ion MS imaging of lipids in picoliter vials with a buckminsterfullerene ion source. Analytical Chemistry 77;2005:6190–6196. [PubMed]
Two papers describe prospects for mass spectral imaging of the distribution of small molecules in cells or tissues with very high spatial resolution. The methodology in both cases is secondary ion time-of-flight mass spectrometry. Touboul et al. use primary ion beams of bismuth clusters, Bi3+ or Bi52+, which provide characteristics better than the more conventional gold clusters. Ions of m/z 23–750 are desorbed from rat brain sections with good yield and possible spatial resolution of 400 nm. The tissue does not require washing or coating, thus avoiding the degradation of spatial resolution that results from such procedures. Ostrowski et al. use buckminsterfullerene (C60+) as the primary ion beam to study lipids. This primary ion provides analyte yields greatly improved over Ga+ or In+ primary ions. Phophatidylethanolamine, phosphatidylglycerol, phophatidylserine, phosphatidylinositol, cholesterol, and sulfatide are shown to be identifiable, and their distribution is imaged with micrometer resolution in a model system of picoliter vials etched in silicon. The capability of imaging sub-cellular distribution of lipids is anticipated. The spatial resolution achieved by secondary ion mass spectrometry in these studies is greatly superior to that so far documented for MALDI, although the mass range is less broad.
Schroeder MJ, Webb DJ, Shabanowitz J, Horowitz AF, Hunt DF. Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry. Journal of Proteome Research 4;2005:1832–1841. [PubMed]
A suite of methods for highly efficient characterization of phosphorlyation sites on a protein of interest is described in the context of a study of the focal adhesion protein, paxillin. The methods include incubation of cells with phosphatase inhibitors prior to lysis, generation of tryptic peptides from the protein in situ on the beads used for immunoprecipitation of the protein from the lysate (peptides from the antibody do not interfere with subsequent analysis), and enrichment of phosphopeptides by methyl esterification and IMAC. Twenty-nine phophorylation sites are reported, 19 of them novel. Additionally, 10 proteins representing other members of focal adhesion complexes are detected as co-precipitants with paxillin during the immunochemical purification.
Torres MP, Thapar R, Marzluff WF, Borchers CH. Phosphatase-directed phosphorylation-site determination: A synthesis of methods for the detection and identification of phosphopeptides. Journal of Proteome Research 4;2005:1628–1635. [PubMed]
This work combines a set of methods for detection of phosphopeptides in a way that minimizes chromatographic separation steps and thus minimizes the potential sample losses those steps entail. IMAC is used to enrich putative phosphopeptides from a tryptic digest of a phosphoprotein. The IMAC beads are then transferred to a MALDI target, and are subjected to phosphatase digestion directly on the target to improve peptide ionization efficiency and the information content of the resulting MS/MS spectra. To determine the sites of phosphorylation within the peptide sequences thus assigned, a list of the m/z values for all possible phosphorylation states and all possible charge states for each of the peptides is compiled, and the list is employed to direct MALDI-MS/MS analysis of the phosphorylated peptides bound to IMAC beads.
Gundry RL, Edward R, Kole TP, Sutton C, Cotter RJ. Disposable hydrophobic surface on MALDI targets for enhancing MS and MS/MS data of peptides. Analytical Chemistry 77;2005:6609–6617. [PubMed]
Wei H, Dean SL, Parkin MC, Nolkrantz K, O’Callaghan JP, Kennedy RT. Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS. Journal of Mass Spectrometry 40;2005:1338–1346. [PubMed]
These two papers present new methods for applying hydrophobic surfaces to MALDI targets in order to reduce sample spot diameters and hence increase sensitivities. Gundry et al. spray-coat the target surface with SILCLEAN 3700, a chemical additive for “self-cleaning” glass and paint. The method is uncomplicated and inexpensive, and the surface is removable and free from memory effects. The coating is patterned to provide hydrophilic holes where the samples are to be spotted. This is accomplished by protecting the sample locations from the spray by the presence of drops of water or ammonium per-sulfate solution. Wei et al. have a more complicated method, in which a MALDI target is sputter coated with gold and then modified with an octadecanethiol self-assembled monolayer. The surface is patterned with hydrophilic holes by using a microcontact printing procedure. However, the samples can be concentrated into spots that as small as 200–500 μm in diameter. Samples are applied in small volumes from nanocolumns—reverse-phase traps with bed volumes of 50–100 nL poured in GELoader tips.
Gillar M, Ollivova P, Daly AE, Gebler JC. Orthogonality of separation in two-dimensional liquid chromatography. Analytical Chemistry 77;2005:6426–6434. [PubMed]
Proteomic studies that prepare tryptic peptides for mass spectrometric analysis by “multidimensional” chromatographic separation usually employ strong cation exchange (SCX) followed by reverse-phase (RP) chromatography for the separation steps. The selectivity of these chromatographic modes is substantially different, so they complement one another well: their “orthogonality” is said to be high. This paper considers the factors that maximize orthogonality, and considers the efficiency of 2D separations in terms of separation selectivity and peak capacity (number of species that are separated). The authors point out that the benefits of a highly efficient separation in the first dimension are realized only when a large number of fractions are collected, although proliferation of fractions reduces sample throughput. A protocol based on successive reverse-phase separations at different pH values is shown to have an orthogonality comparable to SCX followed by RP, yet it has the higher peak capacity (the highest practical peak capacity of the various 2D systems considered), showing this protocol to be advantageous when maximal resolution is desired.
Wang D, Xu W, McGrath SC, Patterson C, Neckers L, Cotter RJ. Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry. Journal of Proteome Research 4;2005:1554–1560. [PubMed]
Sites of ubiquitination are tagged to facilitate their identification by MALDI mass spectrometry. The ubiquitin-conjugated protein is guanidinylated to convert lysine residues to homoarginine, and then digested with trypsin. Because ubiquitin is conjugated to lysine residues on target proteins by iso-peptide bridging through the α-carboxyl group of its C-terminal sequence, Lys-Gly-Gly, digestion leaves a diglycine branch attached to the conjugated lysine on the target peptide. Following digestion, the peptides are N-sulfonated. Sulfonation produces a characteristic fragmentation pattern in the mass spectrometer, and peptides containing a ubiquitination site are clearly identified by the presence of two such groups, one on the peptide N-terminus and the other on the diglycine moiety derived from ubiquitin. The guanidination reaction is performed to block unconjugated lysine residues from sulfonation. Guanidination is performed before trypsin digestion because, although it is selective for lysine side chains, it also blocks the α-amino group of peptides with N-terminal glycine, and must be prevented from blocking the diglycine moiety from ubiquitin in this way.
Nam J-M, Wise AR, Groves JT. Colorimetric bio-barcode amplification assay for cytokines. Analytical Chemistry 77;2005:6985–6988. [PubMed]
This paper describes a bio-barcode detection scheme that avoids complex and expensive steps such as immobilization of oligonucleotides on glass chips with a microarrayer, silver enhancement of immobilized gold nanoparticles on a chip, and light-scattering measurements. The key advance is the development of a barcode probe based on porous silica beads that bear greatly enhanced numbers of barcode DNA probe molecules per particle, allowing for the detection of barcode DNA in simplified ways without loss of sensitivity. In this method, probe particles, which also contain an antibody against the target protein molecule, become cross-linked through the target protein to antibody-linked magnetic beads. Barcode DNA hybridized to these complexes is released from the captured complexes and then quantitated colorimetrically on reverse-phase thin-layer chromatography plates by measuring the red color created by the DNA-mediated aggregation of gold nanoparticles functionalized for recapture of the barcode DNA. The assay is applied to the detection of IL-2 at concentrations down to 30 aM. The approach opens the possibility of point-of-care application requiring only probe solutions and TLC plates.
Stoevesandt O, Köhler K, Fischer R, Johnston CD, Brock R. One-step analysis of protein complexes in microliters of cell lysate. Nature Methods 2;2005:833–835. [PubMed]
Detecting molecular interactions in crude cell lysates conventionally relies on copurification during molecular separation schemes, or on fluorescence correlation spectroscopy (FCS) or fluorescence cross-correlation spectroscopy (FCCS). The fluorescent methods usually involve the use of fluorescent fusion proteins or chemical labeling, and thus require a dedicated experimental design entailing restriction to detecting selected interactions. The method described in this paper uses FCS and FCCS for detecting interactions between unlabeled proteins in crude cell lysates. Pairs of primary antibodies against the interacting partners are added along with secondary antibodies tagged for FCS or FCCS applications, all in one step. The method is suitable for screening compounds for their ability to interfere with selected interactions that depend on signal-induced posttranslational modifications occurring in their physiological context.
Brouillard F, Bensalem N, Hinzpeter A, Tondelier D, Trudel S, Gruber AD, Ollero M, Edelman A. Blue native/SDS-PAGE analysis reveals reduced expression of the mCICA3 protein in cystic fibrosis knock-out mice. Molecular and Cellular Proteomics 4;2005:1762–1775. [PubMed]
Conventional 2D gel electrophoresis has proved inadequate for proteomic studies in which the inclusion of intrinsic membrane proteins is of importance. For example, ion channels are of low abundance relative to soluble proteins, and their hydrophobic nature and high pI values prevent good resolution by isoelectric focusing. In a proteomic study of changes in the membrane proteins of colonic crypt cells associated with the absence of the cystic fibrosis transmembrane regulator (CFTR) in CFTR knock-out mice, Brouillard et al. Instead use blue native gel electrophoresis and then SDS-PAGE in a two-dimensional separation. In blue native gel electrophoresis, Coomassie Blue G250 is added to the sample and to the electrophoresis buffers to confer a negative charge on proteins, which thus migrate anodally. The method is of particular interest because no denaturant is present, so protein complexes are not disrupted. The expression level of complexes containing mCICA3, a member of a family of calcium-activated chloride channels with a key role in mucus secretion by goblet cells, is found to be reduced in the colons of CFTR knock-out animals.
Hood BL, Darfler MM, Guiel TG, Furusato B, Lucas DA, Ringeisen BR, Sesterhenn IA, Conrads TP, Veenstra TD, Krizman DB. Proteomic analysis of formalin-fixed prostate cancer tissue. Molecular and Cellular Proteomics 4;2005:1741–1753. [PubMed]
Formalin fixation and paraffin embedding are standard practices for preserving and storing tissue in clinical laboratories. Tissue samples preserved in this way have hitherto been thought to be beyond proteomic analysis. However, methods for recovering material from such samples are described here. To remove the paraffin, tissue sections of 10-μm thickness are heated at 58°C for 60 min and then washed in SubX organic solvent (Surgipath Medical Industries, Richmond, IL). Following rehydration, the sections are stained with Mayer’s hematoxylin, dehydrated, and then rehydrated with 50% glycerol in water prior to laser-capture microdissection to recover cell types of interest. Protein extracts from the resulting preparations are digested with trypsin for LC/MS/MS or protein microarray analysis, and peptide assignments and protein identifications are made.
Zybailov B, Coleman MK, Florens L, Washburn MP. Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Analytical Chemistry 77;2005:6218–6224. [PubMed]
Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, Resing KA, Ahn NG. Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Molecular and Cellular Protoemics 4;2005:1487–1502.
It has long been recognized that in multidimensional protein identification technology (MudPIT), larger numbers of MS/MS scans are assigned to peptides from proteins of higher abundance than to peptides from proteins of lower abundance. The two papers cited here explore this relationship, and conclude that enumeration of the MS/MS scans assigned to a given protein from different cell samples can be used to detect differences in the expression level of that protein in the samples. Zybailov et al. use this “spectrum counting” approach to assess changes in the expression of membrane proteins (solubilized with an acid-cleavable detergent) between yeast cells grown in rich medium and cells grown in minimal medium in which 15N ammonium sulfate is the sole nitrogen source. Expression levels in this experiment can be assessed both by spectrum counting and by measuring the relative intensities of precursor ion signals for peptides from the two cell cultures. A strong correlation between the two kinds of measurement is observed. Moreover, spectrum counting shows better reproducibility and a wider dynamic range. Old et al. spike varying quantities of standard proteins into mammalian cell extracts and observe strong correlation between MS signal strength for tryptic peptides and numbers of peptides assigned to each protein. Spectrum counting is observed to be the more sensitive method for detecting abundance change.
Wang Y, Rudnick PA, Evans EL, Li J, Zhuang Z, DeVoe DL, Lee CS, Bagley BM. Proteome analysis of micro-dissected tumor tissue using a capillary isoelectric focusing–based multidimensional separation platform coupled with ESI-tandem MS. Analytical Chemistry 77;2005:6549–6556. [PubMed]
Moritz RL, Simpson RJ. Liquid-based free-flow electrophoresis–reversed phase HPLC: A proteomic tool. Nature Methods 2;2005:863–873. [PubMed]
These papers illustrate the advantages of liquid-phase isoelectric focusing as a fractionation method in proteomic analyses. Wang et al. couple capillary isoelectric focusing with reverse-phase chromatography to separate tryptic peptides. They use this methodology to analyze proteins in cells collected by laser capture microdissection of a glioblastoma, and report extensive proteomic data acquired from only 10 μg of total protein, equivalent to 20,000 selectively isolated tumor cells. Moritz and Simpson couple a free-flow isoelectric focusing method with reverse-phase liquid chromatography for preparative separation of intact proteins. In the isoelectric focusing step, a protein mixture is continuously injected into a solution of carrier ampholytes flowing as a thin laminar film between two parallel glass plates. An electric field perpendicular to the direction of flow separates the proteins according to pI value, and the separated proteins are swept into a 96-well microtiter plate by a counter-flow solution as they emerge from the electrode compartment. This system is anticipated to be a useful tool for exploration of low-molecular-weight peptides in cells and tissues.
Capodieci P, Donovan M, Buchinsky H, Jeffers Y, Cordon-Cardo C, Gerald W, Edelson J, Shenoy SM, Singer RH. Gene expression in single cells within tissue. Nature Methods 2;2005:663–665. [PubMed]
A fluorescent in situ hybridization (FISH) technique is described for use on formalin-fixed, paraffin-embedded tissue sections. The method is used for multiplexed analysis of RNA expression in single cells, and applied to the ascertainment of gene expression signatures in epithelial cells with varying pathological morphologies in prostate tumor and benign cells from individual patient samples. The methodology works on tissue sections previously stained with hematoxylin and eosin. The correlation of the FISH data with morphological features documented in this way indicates that the expression patterns can be used to classify the tumor cells in patient samples. The ability to measure multiplexed gene expression in samples that retain their tissue morphology opens the possibility of extending tumor grading procedures and identifying differentiation states.
Gustavsson N, Greber B, Kreitler T, Himmelbauer H, Lehrach H, Gobom J. A proteomic method for the analysis of changes in protein concentrations in response to systematic perturbations using metabolic incorporation of stable isotopes and mass spectrometry. Proteomics 5;2005:3563–3570. [PubMed]
The methodology presented in this paper allows proteomic changes that arise in response to changing cell states to be ascribed to alterations in protein synthesis, degradation, or post-translational modification. It works by coupling an experimental perturbation in a cell culture with a replacement of the growth medium by one enriched in 15N. The relative abundance of 15N and 14N in proteins is then measured by mass spectrometry as a function of time after the perturbation. Changes in rates of synthesis and degradation of individual protein species separated by 2D gel electrophoresis are measured by comparing perturbed and unperturbed cultures following the change in growth medium. The method is applied to the investigation of heat stress in HeLa cells. The rise in expression of some heat shock proteins is ascribed to increased synthesis rate and of others to decreased degradation rate. One protein, vimentin, which is detected only after prolonged heat stress, appears to be a result of post-translational recruitment from an existing pool, possibly by disassembly of intermediate filaments.
Evans MJ, Saghatelian A, Sorensen EJ, Cravatt BF. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nature Biotechnology 23;2005:1303–1307.
In most cases, the protein targets of small bioactive molecules are unknown. This impedes understanding of the mechanisms of action of these molecules and the refinement of their structures for the design of drugs. Among the reasons for the difficulty are the relatively weak and reversible interactions displayed by many small molecules with their targets, and the requirement some interactions display for conditions in intact cells not preserved in cell extracts. The approach used here to overcome these technical problems involves cell-based screens employing libraries of compounds that exert their effects by covalent modification of proteins. A compound that inhibits proliferation of breast cancer cells is shown to target the glycolytic enzyme phosphoglycerate mutase 1 (PGM1). This observation underscores the importance of glycolysis for cancer cell viability and focuses attention on PGM1 as a potential therapeutic target. The methodology provides a promising general approach for chemical genomic screening.
Washietl S, Hofacker IL, Lukasser M, Hüttenhofer A, Stadler PF. Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome. Nature Biotechnology 23;2005:1383–1390.
Diverse experimental evidence now exists that a substantial fraction of the genome is transcribed and that a large fraction of transcribed sequences are not translated. What proportion of these noncoding RNAs (ncRNAs) represents functional RNA species, and what represents “transcriptional noise”? In comparative screens, many known functional ncRNAs (e.g., ribosomal RNAs, transfer RNAs, small nuclear RNAs, small nucleolar RNAs, and RNA components of RNAseP and the signal recognition particle) display compensatory mutations that replace one type of base pair with another in paired regions of the molecule. There are also differences in sequence variation between loop regions and paired regions. These signatures are exploited in this survey to identify conserved noncoding DNA sequences that may produce functional ncRNAs. More than 30,000 such structured RNA elements in the human genome are predicted in this study. About one-third of these elements are located in introns of known protein-coding genes and may play regulatory roles in mRNA splicing. A sixth are potential cis-acting regulatory elements of protein-coding mRNAs. About a half of the detected structures are in intergenic regions at least 10 kb away from any known protein-coding gene, and may represent independent, functional ncRNAs. Most are novel. However, over 40% of the predicted structured RNAs coincide with sites of transcription detected in tiling array studies. It is noteworthy that the number of putative ncRNAs in vertebrate genomes is comparable to that of the protein-coding genes.