Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.
High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps.
We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment.
Application of this method allows whole genome studies from samples where material or yields are limiting.
Illumina; ChIP-seq; Multiplex; Barcoding; Library preparation
Advances in high-throughput gene expression technologies such as microarrays have transformed our understanding of the molecular mechanisms underlying various types of biological processes and diseases. The Applause™ line of products (3'-Amp, WT-Amp ST, and WT-Amp Plus ST) addresses requirements of gene expression microarray users with high-quality RNA (50 nanograms) that want a low-cost, single-day, and reliable sample preparation solution. A comparison was made between the Applause line of amplification products and available vendor data for the GeneChip® WT cDNA Synthesis and Amplification and GeneChip3' IVT Express products. Data generated from 50 ng HeLa, MAQC A, and MAQC B RNA using the Applause 3'-Amp kit were compared to equivalent reported vendor data for the GeneChip 3' IVT Express amplification kit. NuGEN's Applause 3'-Amp outperformed the GeneChip 3' IVT Express with amplifications and labeling completed within 6 hours. It also did better than the GeneChip 3' IVT Express platform on HGU133 Plus 2.0 by percent Present Calls (%P). Differential expression analysis of MAQC samples demonstrated high correlation between the Applause 3'-Amp array data and data generated by quantitative PCR (qPCR) for the MAQC Project (Nature Biotech, 24:1151-1161 (2006)), with an R value of 0.96. Data generated from 50 ng brain and skeletal muscle RNA with the Applause WT-Amp ST and WT-Amp Plus ST kits were compared to equivalent 1 μg vendor data for GeneChip WT cDNA Synthesis and Amplification Kit. In addition to the improved speed (9 hours), the Applause WT-Amp kits also showed better QC array metrics (Pos-vs-Neg AUC, All-Probe-Set-Mean, All-Probe-Set-RLE-Mean) than the GeneChip WT cDNA Synthesis and Amplification kit. The Applause amplification system provides researchers with a fast, economical, and high-quality solution to everyday microarray needs.
DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct DNA fragmentation, end-polishing, and adaptor-ligation steps; often with significant sample loss and hands-on time. EPICENTRE Biotechnologies has developed Nextera™ technology which combines these distinct steps into a single reaction; providing a streamlined, efficient, and high-throughput method for generating bar-coded sequencing libraries from nanogram amounts of DNA, in less than two hours. In vitro transposition with Nextera Transposomes simultaneously fragments and covalently tags the target DNA. Di-tagged sequencing libraries compatible with multiple sequencing platforms can be enriched and bar-coded using suppression PCR. Deep sequencing of Nextera libraries yields sequence depth, coverage, and accuracy that is comparable to control libraries produced using nebulization and standard protocols.
We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.
Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>109 transformants in Escherichia coli and 105-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.
Transplex Whole Transcriptome Amplification (WTA2)a exponentially amplifies RNA producing a double-stranded cDNA library while precisely maintaining differential levels of individual transcripts in test and reference samples. Though originally designed to amplify nanogram quantities of RNA, Transplex WTA2 has been shown to be exceedingly effective for amplification from damaged RNA template (FFPE and laser captured tissue samples) and single-cell input quantities (picograms). The efficacy of Transplex WTA2 amplification for downstream applications, primarily qPCR and expression microarray analysis, is well-documented. It follows that the utilization of next-generation sequencing for gene expression research and diagnostics would be well served by Transplex amplification of RNA isolated from samples of severely restricted quantity or quality. Strategies for the integration of Transplex WTA2 with next-generation sequencing are examined, with particular emphasis on elimination of the characteristic fixed primer sequence associated with each amplicon in the amplification library. Removal of these sites will allow direct entry of the resulting product into the sequencing workflow. Methods under consideration will enable the WTA2 amplicon to feed into the current sample prep protocols for the Illumina GA and GAII, SoLiD 5500/5500xl, and Roche-454 GS FLX/Junior platforms.
The quantitative PCR (QPCR) assay for DNA damage and repair has been used extensively in laboratory species. More recently, it has been adapted to ecological settings. The purpose of this article is to provide a detailed methodological guide that will facilitate its adaptation to additional species, highlight its potential for ecotoxicological and biomonitoring work, and critically review the strengths and limitations of this assay. Major strengths of the assay include very low (nanogram to picogram) amounts of input DNA; direct comparison of damage and repair in the nuclear and mitochondrial genomes, and different parts of the nuclear genome; detection of a wide range of types of DNA damage; very good reproducibility and quantification; applicability to properly preserved frozen samples; simultaneous monitoring of relative mitochondrial genome copy number; and easy adaptation to most species. Potential limitations include the limit of detection (~1 lesion per 105 bases); the inability to distinguish different types of DNA damage; and the need to base quantification of damage on a control or reference sample. I suggest that the QPCR assay is particularly powerful for some ecotoxicological studies.
Electronic supplementary material
The online version of this article (doi:10.1007/s10646-009-0457-4) contains supplementary material, which is available to authorized users.
Mitochondrial DNA; DNA damage; DNA repair; Genotoxicity; Biomarker
Gene expression profiling of small numbers of cells requires high-fidelity amplification of sub-nanogram amounts of RNA. Several methods for RNA amplification are available; however, there has been little consideration of the accuracy of these methods when working with very low-input quantities of RNA as is often required with rare clinical samples. Starting with 250 picograms-3.3 nanograms of total RNA, we compared two linear amplification methods 1) modified T7 and 2) Arcturus RiboAmp HS and a logarithmic amplification, 3) Balanced PCR. Microarray data from each amplification method were validated against quantitative real-time PCR (QPCR) for 37 genes.
For high intensity spots, mean Pearson correlations were quite acceptable for both total RNA and low-input quantities amplified with each of the 3 methods. Microarray filtering and data processing has an important effect on the correlation coefficient results generated by each method. Arrays derived from total RNA had higher Pearson's correlations than did arrays derived from amplified RNA when considering the entire unprocessed dataset, however, when considering a gene set of high signal intensity, the amplified arrays had superior correlation coefficients than did the total RNA arrays.
Gene expression arrays can be obtained with sub-nanogram input of total RNA. High intensity spots showed better correlation on array-array analysis than did unfiltered data, however, QPCR validated the accuracy of gene expression array profiling from low-input quantities of RNA with all 3 amplification techniques. RNA amplification and expression analysis at the sub-nanogram input level is both feasible and accurate if data processing is used to focus attention to high intensity genes for microarrays or if QPCR is used as a gold standard for validation.
We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6 ng, 30 ng, 120 ng, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion of heat- or urea-denatured proteins) for 6 ng to 300 ng RAW 264.7 cell protein analysis in terms of number of peptides and proteins identified. The use of accurate mass and time (AMT) tags resulted in the identification of an additional 16 proteins based on 20 peptides from the 6 ng cell lysate prepared with our approach. When AMT analysis was performed for the 6 ng cell lysate prepared with traditional methods, no reasonable peptide signal could be obtained. In all cases, roughly ~30% of the digested sample was taken for analysis, corresponding to the analysis of a 2 ng aliquot of homogenate from the 6 ng cell lysate.
Several isolates of Salmonella were examined for the capacity to synthesize and release a cholera toxin-like toxin that exerted a biological effect on Chinese hamster ovary cells. Measurements of this Salmonella toxin, which was contained in cell sonic extracts and culture filtrates, were expressed in cholera toxin equivalents (nanograms), since the Chinese hamster ovary cell responses of the cholera toxin and the Salmonella toxin were indistinguishable. Comparative titrations of Salmonella preparations were also performed by using an enzyme-linked immunosorbent assay specific for cholera toxin antigen. The amount of Salmonella toxin synthesized was low (nanogram levels), but the toxin was detectable in cell sonic extracts as early as 6 h after culture inoculation and reached maximal levels by 12 h. Salmonella toxin antigen was not detectable in control culture filtrates until 48 h, but the addition of mitomycin C at 8.5 h resulted in the sudden appearance of toxin antigen at 10 to 12 h, and the toxin antigen level reached a maximum at 14 h. A large peak of Chinese hamster ovary cell activity was observed at 48 h in the control culture, but significant Chinese hamster ovary cell activity was detected as early as 14 h. A larger amount of Chinese hamster ovary cell-reactive material was observed as early as 10 h in cultures grown with mitomycin C. The mechanism of the mitomycin-mediated phenomenon that yielded more toxin in culture filtrates was associated with bacteriophage induction. A bacteriophage plaque assay with a susceptible Salmonella strain revealed that there were free bacteriophage in mitomycin-treated culture filtrates (but not control culture filtrates) at 12 h. Toxin production was greatest when cultures were grown at 30 to 37°C and lowest when cultures were grown at 25°C. The inoculum size and degree of culture aeration (agitation) had little effect on synthesis of the toxin, and toxin production occurred during anaerobic growth.
The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.
Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.
Expression profiling of clinically obtainable tumor specimens has been hindered by the need for microgram quantities of RNA. In vitro transcription (IVT)-based amplifications are most commonly used to amplify small quantities of RNA for microarray analysis. However, significant drawbacks exist with IVT-based amplification, and the need for alternative amplification methods remains. Herein, we validate whole transcriptome amplification (WTA), an exponential amplification technique that produces cDNA libraries and amplified target in 3 to 4 hours from nanogram quantities of total RNA using a combination of cDNA microarrays and quantitative polymerase chain reaction (PCR). We demonstrate that WTA material can serve as a “molecular archive” because a WTA cDNA library can be faithfully amplified through multiple rounds of PCR amplification, allowing it to serve as a bankable and distributable resource. To demonstrate applicability, WTA was combined with laser capture microdissection to profile frozen prostate tissues. Unlike most IVT-based and exponential amplification techniques, WTA does not depend on the presence of a poly-A tail. Thus, we demonstrate that WTA is compatible with artificially degraded RNA and RNA isolated from formalin-fixed paraffin-embedded tissues. Taken together, WTA represents a versatile approach to profile and archive cDNA from minute tumor samples and is compatible with partially degraded RNA.
Exponential RNA amplification; whole transcriptome amplification; prostate cancer; laser capture microdissection; FFPE tissue
Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.
Solution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible.
We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company.
This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.
Targeted genomic enrichment; Sequence capture; Massively parallel sequencing; Illumina
The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS.
We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data.
In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads.
By providing sequence data on millions of short DNA fragments in parallel, Next Generation Sequencing has revolutionized biomedical research. In particular, the technique has enabled RNA expression analysis over the entire transcriptome with high sensitivity and dynamic range. With this has come a drive to utilize smaller sample inputs—with the goal of analyzing the transcriptome of a single cell.
One powerful method for cDNA preparation is SMART™ technology (Switching Mechanism At the 5' end of the RNA Template), which utilizes the template switching activity of reverse transcriptase to enable the direct addition of a PCR adaptor to the 3' end of the first-strand cDNA. The result is a single-tube protocol that enhances library amplification efficiency, while minimizing the chance for contamination, making it ideal for library preparation from small amounts of starting material. Indeed, the SMARTer™ Ultra Low RNA method allows researchers to readily obtain high quality data from as little as 10 pg of total RNA. Recent publications indicate that this technology is effective in single cell analysis.
Here we present data which demonstrates that SMART faithfully produces full-length cDNA for use as the template in library sample preparation. Sequencing results for libraries generated from 0.01 to 10 ng of mouse brain total RNA demonstrate that even with just 10 pg of input RNA, over 90% of the data maps to the genome, and the average transcript coverage is as uniform as that seen with much greater amounts of RNA. Also, under all conditions tested, rRNA reads accounted for only 3–5% of the total reads. Finally, comparing results to quantitative PCR for the MAQC (microarray quality control) gene set shows a high correlation with libraries made with 1 or 0.1 ng RNA. These data indicate that the SMART cDNA preparation method is an ideal choice for single cell transcriptome analysis.
A high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies.
We used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80.
Cultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.
Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.
Enviroxime has been shown to inhibit the replication of rhinoviruses and other enteroviruses in concentrations as low as nanograms per milliliter in in vitro assays but is markedly less effective in clinical trials. The marked hydrophobicity and water insolubility of this compound may be a factor for this disparity. To overcome this handicap, we incorporated enviroxime into liposomes and then tested the antirhinovirus activity and toxicity of the liposome-incorporated enviroxime (LE) in cell culture and studied its administration by small-particle aerosol. Free enviroxime and LE were found to have equivalent efficacies against rhinovirus strains 1A and 13 in in vitro assays; however, preparations of LE were 10- to greater than or equal to 50-fold less toxic to tissue culture cells than was free enviroxime. In contrast to free enviroxime, which could not be delivered by small-particle aerosol because of its water insolubility, LE (4 mg/ml) was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tracts of mice; after just 20 min, significant levels of enviroxime were detected in the lungs and noses of exposed mice. Moreover, mice exposed to aerosols of liposomes containing both enviroxime and fluorescein isothiophosphatidylethanolamine showed accumulations of the fluorescent marker in the lungs, particularly in or around the tall columnar epithelial cells lining the bronchi and bronchioles.
The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.
The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.
The clinical relevance of the amount of human papillomavirus type 18 (HPV18) DNA in cervical tissue (ie, HPV18 DNA load) is unknown.
Study subjects were 303 women who were HPV18 positive at enrollment into the Atypical Squamous Cells of Undetermined Significance (ASC-US) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. HPV18 DNA load, expressed as copies of HPV18 per nanogram of cellular DNA, at enrollment was quantitatively measured. Subjects were followed up semiannually for a period of 2 years for detection of cervical intraepithelial neoplasia 2–3 (CIN2–3). A linear regression model was used to examine associations of CIN2–3 with HPV18 DNA load. All statistical tests were two-sided.
CIN2–3 was confirmed in 92 of 303 (30.4%) HPV18-positive women. Among women without CIN2–3, HPV18 DNA load was positively associated with increasing severity of cervical cytology at enrollment (Ptrend < .001). However, among those with CIN2–3, HPV18 DNA load was not associated with severity of cervical cytology at enrollment (Ptrend = .33). The ratios of geometric means of HPV18 DNA load at enrollment among women with CIN2–3, relative to those without, were 6.06 (95% confidence interval [CI] = 0.31 to 117.92) for those with normal cytology at enrollment, 0.50 (95% CI = 0.10 to 2.44) for those with ASC-US, 0.11 (95% CI = 0.03 to 0.46) for those with LSIL, and 0.07 (95% CI = 0.01 to 0.80) for those with high-grade squamous intraepithelial lesion (HSIL). After adjusting for age and coinfection with other high-risk HPVs, a statistically significant association of lower HPV18 DNA load with CIN2–3 was observed among women with LSIL or HSIL at enrollment (P = .02). Within the 2-year period, HPV18 DNA load was unrelated to the timing of CIN2–3 diagnosis. Overall results were similar when the outcome was CIN3.
HPV18 DNA load was higher for women with LSIL or HSIL at enrollment with no evidence of CIN2–3 during the 2-year follow-up period than it was for women with CIN2–3. Thus, testing for high levels of HPV18 DNA does not appear to be clinically useful.
Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.