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Reliable amplification method for bacterial RNA
Abstract
DNA microarray technology has been increasingly applied for studies of clinical samples. Frequently, RNA probes from clinical samples are available in limited amounts. We describe a reliable amplification method for bacterial RNA. We verified this method on mycobacterial RNA applying mycobacterial genome-directed primers (mtGDPs). Glass slide-based oligoarrays were employed to assess the quality of the amplification method. We observed a relatively small bias in amplified RNA pool when compared to the unamplified one. Up to 1000-fold linear RNA amplification in a single amplification round was obtained. To our knowledge, this study describes the first amplification method for mycobacterial RNA.
... The second priming strategy used a set of mycobacterial amplification-directed primers (with the addition of a T7 promoter sequence) to prime the first strand cDNA synthesis instead of oligo-dT (as in the eukaryotic system) followed by T7 transcript amplification. A minimal set of amplification-directed primers (ADP) was designed to prime all the genes in the combined genomes of M. tuberculosis H37Rv [10], CDC1551 [11], and M. bovis AF2122/ 97 [12] using a process first described by Talaat et al. [13], and which have recently been used in conjunction with a template switching strategy for bacterial RNA amplification [14][15][16]. ...
... ADP primers were designed to initiate cDNA synthesis within 500 nucleotides of the 3' of the coding sequences of each gene, however primers could bind elsewhere in the ORF. Thus ADP priming will initiate cDNA synthesis at multiple points within the message, in contrast oligo-dT priming will only occur within the terminal polyadenylated tails added to the 3' end of the polycistronic bacterial message (or indeed intragenic polyA sequences); this pattern was also observed by Rachman et al. [14]. Therefore ADP primed amplification would be expected to yield a shorter modal length product than oligo-dT primed RNA amplification and this was indeed the case. ...
... This compares to other methods of amplification with correlation coefficients of amplified to unamplified of 0.44-0.75 for inputs of 10-5000 ng bacterial RNA [15]; or 0.73-0.99 for inputs of 10-100 ng [14]. Despite these relatively high correlation coefficients however, the number of genes over or under-represented in amplified compared to unamplified profiles for the same source RNA gives a better indication of method biasing. ...
The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers both linked to T7 polymerase in vitro run off transcription.
The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5 ng total M. tuberculosis RNA with unamplified RNA from the same source. In addition, as a direct measure of the effectiveness of bacterial amplification for identifying biologically relevant changes in gene expression, a model M. tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5 ng total RNA (~equivalent to 2 x 105 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles using both methods.
Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling.
... BCG (Pasteur 1173P2) was kindly provided by the Pasteur Institute (Paris, France). The mycobacterial strains used in this study were cultured as described previously 59 . Total RNA was isolated from Mtb H37Rv and K strains grown to an optical density of OD 600 = 0.5. ...
... Kaufmann (Max Planck Institute for Infection Biology, Berlin, Germany). RNA labelling and array hybridisation were performed as described previously 59 . The hybridised slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, CA, USA), and spot intensities were identified and quantified with the TM4 Microarray Software Suite (http://www.tm4.org). ...
Accumulating evidence indicates that latency-associated Mycobacterium tuberculosis (Mtb)-specific antigens from the dormancy survival regulator regulon (DosR) may be promising novel vaccine target antigens for the development of an improved tuberculosis vaccine. After transcriptional profiling of DosR-related genes in the hyper-virulent Beijing Mtb strain K and the reference Mtb strain H37Rv, we selected Rv3131, a hypothetical nitroreductase, as a vaccine antigen and evaluated its vaccine efficacy against Mtb K. Mtb K exhibited stable and constitutive up-regulation of rv3131 relative to Mtb H37Rv under three different growth conditions (at least 2-fold induction) including exponential growth in normal culture conditions, hypoxia, and inside macrophages. Mice immunised with Rv3131 formulated in GLA-SE, a well-defined TLR4 adjuvant, displayed enhanced Rv3131-specific IFN-γ and serum IgG2c responses along with effector/memory T cell expansion and remarkable generation of Rv3131-specific multifunctional CD4+ T cells co-producing TNF-α, IFN-γ and IL-2 in both spleen and lung. Following challenge with Mtb K, the Rv3131/GLA-SE-immunised group exhibited a significant reduction in bacterial number and less extensive lung inflammation accompanied by the obvious persistence of Rv3131-specific multifunctional CD4+ T cells. These results suggest that Rv3131 could be an excellent candidate for potential use in a multi-antigenic Mtb subunit vaccine, especially against Mtb Beijing strains.
... For the 1-year sample, an additional amplification step was required to obtain sufficient quantities of RNA as described previously (Rachman et al., 2006). Amplification comprised two steps: double-stranded cDNA synthesis was followed by in vitro RNA amplification. ...
... Subsequently, columns were dried by centrifugation at 8,000×g and 20°C for 2 min and RNAs were eluted with 50 µl RNase-free water per column. For the 1-year sample, an additional amplification step was required to obtain sufficient quantities of RNA as described previously (Rachman et al., 2006). Amplification comprised two steps: double-stranded cDNA synthesis was followed by in vitro RNA amplification. ...
... Ten years ago, this technique was quantitatively evaluated by microarray analysis (5,102,132,137) and modified in many ways, including T7 RNA polymerase-based linear RNA amplification (in vitro transcription, IVT) (79,83,151), PCR-based exponential strategy (51,56), and linear isothermal amplification of cDNA using a single primer (21,111); however, all of these methods were originally designed for a study using eukaryotic mRNA, which possesses a poly(A) tail at its 3'-end. To amplify prokaryotic RNA, a linear amplification strategy based on T7 RNA polymerase was developed (61,96). This method uses the overhang tailing activity of the Moloney murine leukemia virus reverse transcriptase to add additional nontemplate residues, normally cytosines, to the 3'-end of the first strand of cDNA. ...
... To avoid laborious RNA extraction from soil and high costs for many purification columns, whole transcriptome RNA amplification is an alternative method of obtaining large amounts of RNA (142). Although several amplification methods have been reported (36,61,81,96), it is more convenient to use a commercially available kit. To the best of our knowledge, until now, only one commercially available kit has been designed for the amplification of prokaryotic RNA, the MessageAmp II-Bacteria Kit (Ambion). ...
Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.
... Mtb RNA (2 μg) was enzymatically labelled with Cy3 fluorophore and hybridised to a Mtb complex microarray (ArrayExpress accession number A-BUGS-41) as previously described (Salina et al., 2014). For quantitative RT-PCR, total RNA was isolated from triplicate Mtb cultures; cDNA was generated using Superscript Reverse Transcriptase II and mycobacterial genome-directed primers (Rachman et al., 2006). qPCR was performed in a Corbett Rotor Gene 6000 real-time thermocycler using Absolute qPCR SYBR Green mix and gene expression values were normalised to 16S rRNA expression. ...
Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi‐drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes, that are likely caused by reduced phosphorylation of the H‐NS‐like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H‐NS, Lsr2 binds DNA in sequence‐dependent and non‐specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP‐sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2. Protein kinase B (PknB) is essential for growth of Mycobacterium tuberculosis because of its central role in regulation of peptidoglycan biosynthesis. Here we describe that PknB, controls activation of alternative pathways that are not required for active growth but crucial for survival in stressful environment and virulence. PknB phosphorylates Lsr2, a global transcriptional regulator, at threonine 112 and controls its binding to DNA and transcription of target genes.
... The Mtb K strain was obtained from the strain collection at the Korean Institute of Tuberculosis (Osong, Chungchungbuk-do, Korea). All of the mycobacterial strains used in this study were cultured and prepared as described previously (Rachman et al., 2006). ...
In a previous study, we have identified MTBK_24820, the complete protein form of PPE39 in the hypervirulent Mycobacterium tuberculosis (Mtb) strain Beijing/K by using comparative genomic analysis. PPE39 exhibited vaccine potential against Mtb challenge in a murine model. Thus, in this present study, we characterize PPE39-induced immunological features by investigating the interaction of PPE39 with dendritic cells (DCs). PPE39-treated DCs display reduced dextran uptake and enhanced MHC-I, MHC-II, CD80 and CD86 expression, indicating that this PPE protein induces phenotypic DC maturation. In addition, PPE39-treated DCs produce TNF-α, IL-6 and IL-12p70 to a similar and/or greater extent than lipopolysaccharide-treated DCs in a dose-dependent manner. The activating effect of PPE39 on DCs was mediated by TLR4 through downstream MAPK and NF-κB signaling pathways. Moreover, PPE39-treated DCs promoted naïve CD4+ T-cell proliferation accompanied by remarkable increases of IFN-γ and IL-2 secretion levels, and an increase in the Th1-related transcription factor T-bet but not in Th2-associated expression of GATA-3, suggesting that PPE39 induces Th1-type T-cell responses through DC activation. Collectively, the results indicate that the complete form of PPE39 is a so-far-unknown TLR4 agonist that induces Th1-cell biased immune responses by interacting with DCs.
This article has an associated First Person interview with the first author of the paper.
... BCG (Pasteur 1173P2) was kindly provided by Dr. Brosch from the Pasteur Institute (Paris, France). All Mtb strains used in the current study were cultured and prepared as previously described in Han et al. (33). ...
Bacillus Calmette‐Guerin vaccine confers insufficient pulmonary protection against tuberculosis (TB), particularly the Mycobacterium tuberculosis (Mtb) Beijing strain infection. Identification of vaccine antigens (Ags) by considering Mtb genetic diversity is crucial for the development of improved TB vaccine. MTBK_20640, a new Beijing genotype‐specific proline‐glutamic acid–family Ag, was identified by comparative genomic analysis. Its immunologic features were characterized by evaluating interactions with dendritic cells (DCs), and immunogenicity and vaccine efficacy were determined against highly virulent Mtb Beijing outbreak Korean Beijing (K) strain and HN878 strain in murine infection model. MTBK_20640 induced DCs via TLR2 and downstream MAPK and NF‐κB signaling pathways, effectively promoting naive CD4‐positive (CD4⁺) T‐cell proliferation and IFN‐γ production. Different IFN‐γ response was observed in mice infected with Mtb K or reference H37Rv strain. Significant induction of T helper type 1 cell‐polarized Ag‐specific multifunctional CD4⁺ T cells and a marked Ag‐specific IgG2c response were observed in mice immunized with MTBK_20640/glucopyranosyl lipid adjuvant‐stable emulsion. The immunization conferred long‐term protection against 2 Mtb Beijing outbreak strains, as evidenced by a significant reduction in colony‐forming units in the lung and spleen and reduced lung inflammation. MTBK_20640 vaccination conferred long‐term protection against highly virulent Mtb Beijing strains. MTBK_20640 may be developed into a novel Ag component in multisubunit TB vaccines in the future.—Kwon, K. W., Choi, H.‐H., Han, S. J., Kim, J.‐S., Kim, W. S., Kim, H., Kim, L.‐H., Kang, S. M., Park, J., Shin, S. J. Vaccine efficacy of a Mycobacterium tuberculosis Beijing‐specific proline‐glutamic acid (PE) antigen against highly virulent outbreak isolates. FASEB J. 33, 6483–6496 (2019). www.fasebj.org
... BCG (Pasteur 1173P2) was kindly provided by the Pasteur Institute (Paris, France). The mycobacterial strains used in this study were cultured as described previously 51 . Enumerated mycobacteria were used for subsequent experiments. ...
Antigens (Ags) in Mycobacterium tuberculosis (Mtb) that are constitutively expressed, overexpressed during growth, essential for survival, and highly conserved may be good vaccine targets if they induce the appropriate anti-Mtb Th1 immune response. In this context, stress response-related antigens of Mtb might serve as attractive targets for vaccine development as they are rapidly expressed and are up-regulated during Mtb infection in vivo. Our group recently demonstrated that GrpE, encoded by rv0351 as a cofactor of heat-shock protein 70 (HSP70) in the DnaK operon, is a novel immune activator that interacts with DCs to generate Th1-biased memory T cells in an antigen-specific manner. In this study, GrpE was evaluated as a subunit vaccine in comparison with the well-known HSP70 against the hyper-virulent Mtb Beijing K-strain. Both HSP70- and GrpE-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 in the lung and spleen of Mtb-infected mice, but GrpE only produced a similar level of IFN-γ to that produced by ESAT-6 stimulation during the late phase and the early phase of Mtb K infection, indicating that GrpE is highly-well recognised by the host immune system as a T cell antigen. Mice immunised with the GrpE subunit vaccine displayed enhanced antigen-specific IFN-γ and serum IgG2c responses along with antigen-specific effector/memory T cell expansion in the lungs. In addition, GrpE-immunisation markedly induced multifunctional Th1-type CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs of Mtb K-infected mice, whereas HSP70-immunisation induced mixed Th1/Th2 immune responses. GrpE-immunisation conferred a more significant protective effect than that of HSP70-immunisation in terms of bacterial reduction and improved inflammation, accompanied by the remarkable persistence of GrpE-specific multifunctional CD4+ T cells. These results suggest that GrpE is an excellent vaccine antigen component for the development of a multi-antigenic Mtb subunit vaccine by generating Th1-biased memory T cells with multifunctional capacity, and confers durable protection against the highly virulent Mtb K.
... Sequencing of the M. tuberculosis H37Rv genome [1], and subsequent mouse [33] and human genomes [34], heralded the age of genome-wide expression profiling using microarrays, qRT-PCR panels or more recently RNAseq [35]. These whole genome approaches together with the continued development of mRNA extraction, stabilisation, and amplification methodologies [36][37][38][39][40] have enabled previously intractable scenarios to be investigated, generating rich datasets describing host and pathogen responses to infection. The first studies measuring transcriptional changes in host cells contrasted the gene expression patterns of macrophages after infection with different pathogens. ...
The ability to genome sequence mycobacteria and host organisms has enabled a range of system-wide approaches to be developed to explore the interplay between host and pathogen. These global analyses offer an unbiased means of generating new hypotheses to further understand bacterial pathogenesis and immune activation states. Mycobacterium tuberculosis high-throughput mutant screening has identified key genes and pathways involved in mycobacterial physiology or pathogenicity that are required in vivo or during macrophage infection. Reciprocal genome-wide RNAi-based screening approaches have highlighted host genes that play crucial roles in the immune and metabolic crosstalk with infecting bacilli. In addition to these loss-of-function screens, transcriptional profiling of the pathogen, of the host, or of both together has provided clues into the divergent metabolic states and key signalling events that characterise M. tuberculosis infection. Such global analyses, linked in a systems approach through interaction databases and network mapping, allow descriptive and predictive models of infection and disease to be constructed. In this chapter we review the recent developments and applications of these system-wide approaches to better understand the interactions of M. tuberculosis with its host.
... RNA Preparation, Transcriptomic, and qRT-PCR Analysis Total RNA from mycobacterial cells was prepared as previously described (Dietrich, 2001) for all samples except the 1-year time point. For the 1-year time point we applied an optimized amplification protocol (Rachman et al., 2006). We validated array results using qRT-PCR (Figure S3 ) and could qualitatively confirm microarray results in approximately 90% of tested cases (40 out of 44 qRT-PCRs with single peaks in the dissociation curves). ...
Attenuated strains of mycobacteria can be exploited to determine genes essential for their pathogenesis and persistence. To this goal, we sequenced the genome of H37Ra, an attenuated variant of Mycobacterium tuberculosis H37Rv strain. Comparison with H37Rv revealed three unique coding region polymorphisms. One polymorphism was located in the DNA-binding domain of the transcriptional regulator PhoP, causing the protein's diminished DNA-binding capacity. Temporal gene expression profiles showed that several genes with reduced expression in H37Ra were also repressed in an H37Rv phoP knockout strain. At later time points, genes of the dormancy regulon, typically expressed in a state of nonreplicating persistence, were upregulated in H37Ra. Complementation of H37Ra with H37Rv phoP partially restored its persistence in a murine macrophage infection model. Our approach demonstrates the feasibility of identifying minute but distinct differences between isogenic strains and illustrates the consequences of single point mutations on the survival stratagem of M. tuberculosis.
Nucleic acids, especially DNA, are targets of qualitative and quantitative diagnostics for genetically modified organisms
(GMO) in seeds, food- and feedstuff. The amplification of the nucleic acid is an essential step for further analyses of the
target sequence. The PCR has been the method of choice for DNA amplification in most laboratories, and its real-time version
(qPCR) also enables quantitative analysis of target contents. Despite its numerous advantages, PCR technology has some limitations
such as the lack of true multiplexing properties. To alleviate the drawbacks linked to PCR technology, alternative nucleic
acid amplification methods with promising characteristics are being developed fast. These methods, their advantages, and the
inconveniences, which are not yet resolved are summarized in the paper. Special focus is given to the possibilities of using
these alternative methods for GMO detection in future, when expansion of GMOs both in diversity and frequencies will make
current GMO detection systems difficult to operate.
Tuberculosis (TB) remains a serious threat to humankind, and humans have encountered the causative agent of TB, Mycobacterium tuberculosis (MTB), for more than 10,000 years. Despite rapid advances in technology, efforts to besiege this robust pathogen seem to fail. The availability of genome sequences of several MTB complex strains open a new era of MTB research, the functional genomics, which will provide guidelines for novel control measures. In recent years, a series of methods have been developed to explore the mechanisms employed by MTB to persist and cause disease in the host. DNA array technology enables us to perform comparative genomics of different MTB strains and to examine the gene expression profiles of MTB growing under diverse living conditions. The generated transcriptome data can be exploited for design of new drugs, especially against multidrug-resistant (MDR) strains, development of more efficient vaccines, and identification of biomarkers for better diagnosis.
Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species—including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition—and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction (≈50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.
• bacterial communities
• metagenomics
• metatranscriptomics
• marine
• cDNA
Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped lambda clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies.
Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected
human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film
(ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide;
a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows
selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain
reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.
Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.
We describe a rapid noncontact method for the capture of single cells or small tissue areas of any size or shape directly within the cap of a common microfuge tube. Prior to the laser-mediated transfer, the specimen is isolated by laser microbeam microdissection, forming a clear-cut gap around the selected area. Laser treatment does not impair subsequent RNA analysis. We have used this method to isolate a single cell from archival colon adenocarcinoma, and were able to detect point mutations within codon 12 of c-Ki-ras2 mRNA after nested RT-PCR analysis.
Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.
A new method for amplifying cDNA ends is described which requires only first-strand cDNA synthesis and a single PCR to generate
a correct product with very low or no background. The method can be successfully applied to total RNA as well as poly A+ RNA.
The same first-strand cDNA can be used to amplify flanking sequences of any cDNA species present in the sample.
Multiple Arabidopsis thaliana clones from an experimental series of cDNA microarrays are evaluated in order to identify essential sources of noise in the
spotting and hybridization process. Theoretical and experimental strategies for an improved quantitative evaluation of cDNA
microarrays are proposed and tested on a series of differently diluted control clones. Several sources of noise are identified
from the data. Systematic and stochastic fluctuations in the spotting process are reduced by control spots and statistical
techniques. The reliability of slide to slide comparison is critically assessed within the statistical framework of pattern
matching and classification.
DNA microarrays have the ability to analyze the expression of thousands of the same set of genes under at least two different experimental conditions. However, DNA microarrays require substantial amounts of RNA to generate the probes, especially when bacterial RNA is used for hybridization (50 microg of bacterial total RNA contains approximately 2 microg of mRNA). We have developed a computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in a given genome. The algorithm predicts, for example, that 37 oligonucleotides should prime all genes in the Mycobacterium tuberculosis genome. We tested the usefulness of the genome-directed primers (GDPs) in comparison to random primers for gene expression profiling using DNA microarrays. Both types of primers were used to generate fluorescent-labeled probes and to hybridize to an array of 960 mycobacterial genes. Compared to random-primer probes, the GDP probes were more sensitive and more specific, especially when mammalian RNA samples were spiked with mycobacterial RNA. The GDPs were used for gene expression profiling of mycobacterial cultures grown to early log or stationary growth phases. This approach could be useful for accurate genome-wide expression analysis, especially for in vivo gene expression profiling, as well as directed amplification of sequenced genomes.
To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide
probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The
specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity
was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide
probes and PCR probes was observed and both had a minimum reproducible detection limit of ∼10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any ‘non-target’ transcripts (cDNAs)
>75% similar over the 50 base target may show cross-hybridization. Thus non-target sequences which have >75–80% sequence similarity
with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity.
In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15
contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue
or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results
validate the utility of oligonucleotide probe (50mer) glass microarrays.
Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.
Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).
Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.
Motivation:
While the use of cDNA microarrays for functional genomic analysis has become commonplace, relatively little attention has been placed on false positives, i.e. the likelihood that a change in measured radioactive or fluorescence intensity may reflect a change in gene expression when, in fact, there is none. Since cDNA arrays are being increasingly used to rapidly distinguish biomarkers for disease detection and subsequent assay development (Wellman et al., Blood, 96, 398-404, 2000), the impact of false positives can be significant. For the use of this technology, it is necessary to develop quantitative criteria for reduction of false positives with radioactively-labeled cDNA arrays.
Results:
We used a single source of RNA (HuT78 T lymphoma cells) to eliminate sample variation and quantitatively examined intensity ratios using radioactively labeled cDNA microarrays. Variation in intensity ratios was reduced by processing microarrays in side-by-side (parallel mode) rather than by using the same microarray for two hybridizations (sequential mode). Based on statistical independence, calculation of the expected number of false positives as a function of threshold showed that a detection limit of [log(2)R] >0.65 with agreement from three replicates could be used to identify up- or down-modulated genes. Using this quantitative criteria, gene expression differences between two related T lymphoma cell lines, HuT78 and H9, were identified. The relevance of these findings to the known functional differences between these cell types is discussed.
Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated. Priming with oligo(dT) yielded more cDNA than priming with an arbitrary primer for only 1 of 12 unrelated M. tuberculosis genes, as measured by competitive PCR. Priming with oligo(dT) yielded cDNA for only 30% of the genes primed for by 37 M. tuberculosis genome-directed oligonucleotides, as assessed by hybridization of cDNA with an M. tuberculosis microarray. These data demonstrate that priming of reverse transcription of mycobacterial mRNA with oligo(dT) does not yield representative samples of cDNA.
The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation
by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing
only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and
degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the
chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich
tails. These heterogeneous tails were found in the amino acid coding region, the 5′ and 3′ untranslated regions of mRNAs,
as well as in rRNA and the single intron located at the tRNAfmet. Furthermore, unlikeE. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and
degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.
Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolateRNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker withthe ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy toperform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazardorganisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primarymacrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which arerather resistant to genetic manipulation, like Mycobacterium bovis. s 2000 Federation of European Microbiological Societies. Publishedby Elsevier Science B.V. All rights reserved.Keywords: Mycobacterium; RNA isolation; In vivo
We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.
[The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.]
This chapter describes one of the currently used microarray technologies commonly called “spotting” or “printing” because DNAs are physically spotted on a solid substrate in which short oligonucleotides is synthesized directly on a solid support. In standard spotting applications, large collections of DNA samples are assembled in 96- or 384-well plates. DNA microarrays are used for a variety of purposes; essentially any property of a DNA sequence that can be made experimentally to result in differential recovery of that sequence can be assayed for thousands of sequences at once by DNA microarray hybridization. The chapter focuses on the application of DNA microarrays to gene expression studies and discusses general principles of whole genome expression monitoring as well as detailing the specific process of making and using spotted DNA microarrays.
The possibility that mRNA from Mycobacterium tuberculosis and M. bovis BCG may present polyadenylation at the 3' end was investigated. The total RNA, extracted from the bacterial cells and treated with DNase, was used as substrate for reverse transcriptase (RT)-dependent cDNA synthesis. The RT reaction was primed with oligo(dT) and with downstream specific primers for the genes of the antigens 65 KDa and 85-C. PCR probing of the reaction products for cDNAs of the two mycobacterial genes yielded the expected 225 and 307 bp bands when RT synthesis was primed by oligo(dT) and by downstream specific primers. Reaction products from oligo(dT)-primed RT of RNase-treated RNA and untranscribed RNA, probed by PCR, failed to generate the 225 and 307 bp specific bands. These findings support the existence of polyadenylated tracts in mRNA of mycobacteria that can be targeted by oligo(dT) primers to initiate RT-dependent cDNA synthesis. This may result in an advance in the study of gene expression in these and possibly other bacteria.
Gene expression microarrays hold great promise for studies of human disease states. There are significant technical issues specific to utilizing clinical tissue samples which have yet to be rigorously addressed and completely overcome. Precise, quantitative measurement of gene expression profiles from specific cell populations is at hand, offering the scientific community the first comprehensive view of the in vivo molecular anatomy of normal cells and their diseased counterparts. Here, we propose a model for integrating-in three dimensions-expression data obtained using the microarray.
To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria.
Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.
Experimental genomics in combination with the growing body of sequence information promise to revolutionize the way cells and cellular processes are studied. Information on genomic sequence can be used experimentally with high-density DNA arrays that allow complex mixtures of RNA and DNA to be interrogated in a parallel and quantitative fashion. DNA arrays can be used for many different purposes, most prominently to measure levels of gene expression (messenger RNA abundance) for tens of thousands of genes simultaneously. Measurements of gene expression and other applications of arrays embody much of what is implied by the term 'genomics'; they are broad in scope, large in scale, and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding.
Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half-lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp-lacZ and rne-lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5' untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.
RNA amplification results in reproducible microarray data with slight ratio bias Oligo(dT)-primed synthesis of cDNA by reverse transcriptase in mycobacteria
- L G Puskas
- A Zvara
- L Hackler Jr
- P Van Hummelen
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Normalization strategies for cDNA microarrays Identification of expressed genes by laser-mediated manipulation of single cells
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Schuchhardt, J., Beule, D., Malik, A., Wolski, E., Eickhoff, H., Lehrach, H., Herzel, H., 2000. Normalization strategies for cDNA microarrays. Nucleic Acids Res. 28, E47. Schutze, K., Lahr, G., 1998. Identification of expressed genes by laser-mediated manipulation of single cells. Nat. Biotechnol. 16, 737–742.
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Genomics, gene expression and DNA arrays
- Lockhart