Picogram Quantities Research Articles

Quantitation of ceramide phosphorylethanolamines containing saturated and unsaturated sphingoid base cores

Sphingomyelin (SM) and ceramide-phosphoethanolamines (cer-PEs) are related lipids present in mammals and insects, respectively. Owing to the critical roles that cer-PEs play in eukaryotic cellular function, there is a need to develop methods that provide accurate quantitation of these compounds. Results obtained in this study demonstrate that Drosophila contains cer-PEs with unsaturated sphingoid base cores as well as low levels of cer-PEs that possess saturated sphingoid base cores. Specifically, the method developed in this study enabled the quantitation of picogram amounts of cer-PE containing both unsaturated d14:1 Δ4 and d16:1 Δ4 and saturated d14:0 sphingoid base cores. Using this method, cer-PE compounds with both saturated and unsaturated sphingoid base cores were initially identified by neutral loss scanning, followed by quantitation using selected reaction monitoring (SRM) scans. The SRM scans measured a product ion originating from the sphingoid base backbone, rather than from the head group, increasing the specificity and sensitivity of the quantitation measurement.

Jumping off the Page

Chemistry![Figure][1] CREDIT: © SCIENCEPHOTOS/ALAMY Few marriages of analytical methods have been as successful as that of chromatography and mass spectrometry; together they can tease out the chemical composition of extraordinarily complex mixtures. A typical apparatus incorporates a gas or liquid chromatograph, in which analytes travel through a separation column, and a downstream detector where their masses are measured. An early, no-frills variant of chromatography involved spotting samples on paper, an inexpensive and highly portable support medium. Wang et al. now show that this simple material can also be used as an ionization platform for introducing samples into mass spectrometers. Samples such as blood are spotted on the paper, which is then cut to a sharp triangular point. The paper is wet with a methanol-water solution, and a high positive bias (4.5 kV) is applied to the paper relative to the nearby inlet of a tandem mass spectrometry under ambient conditions; the precise mechanism for ion release remains somewhat unclear. The authors demonstrate the detection of drugs such as Gleevec in blood, as well as picogram quantities of cocaine swabbed from a surface. Chromatographic methods can also be used to separate components in a sample along the paper, which can then be cut into separate pieces for further analysis. Angew. Chem. Int. Ed. 49 , 10.1002/anie.200906314 (2010). [1]: pending:yes

An Optimized Procedure for DNA Silver Staining in Polyacrylamide Gels

ABSTRACTAn optimized procedure for staining of DNA in polyacrylamide gel electrophoresis was created. It was found that picogram quantities of DNA can be detected in about one hour. The major advantages of the method proposed are to be relatively simple and fast, as well as enough sensitive for some of the most common applications.

Probing magnetic and gold nanoparticles by using MAClevers® as ultrasensitive sensors

Magnetic AFM probes known as MAClevers® were employed for sensing picogram amounts of magnetic nanoparticles, based on the cantilever frequency shifts resulting from the magnetically induced adsorption of mass. By using organothiol functionalized magnetic nanoparticles, this analytical strategy was successfully extended to the detection of gold nanoparticles, as confirmed by confocal Raman microscopy.

Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant

Molecular-grade glycogen is widely used to recover nanogram or picogram quantities of DNA and RNA across molecular biology applications in the life sciences. As a result, its purity is critical to obtain reliable results. Using agarose gel electrophoresis, we detected pg/microL (DNA) to ng/microL (RNA) concentrations of nucleic acid in two of the nine glycogen samples obtained from commercial suppliers. Denaturing gradient gel electrophoresis of 16S rRNA gene PCR-amplified products indicated that an additional two samples contained detectable contamination. We also tested a synthetic polymer co-precipitant, linear polyacrylamide (LPA); none of the four samples tested with LPA were detectably contaminated. The partial 16S rRNA gene sequence associated with the contaminated samples of the shellfish-derived glycogen was nearly identical to the sequence of Actinobacteria lwoffii, which has been isolated from mussels previously. By testing the recovery of low-nanogram amounts of DNA with multiple precipitants and simulated experimental conditions, we demonstrated that LPA was a preferable co-precipitant for sensitive protocols.

A continuous flow mass spectrometry technique of argon isotope measurement for K/Ar geochronology

A new method for the measurement of argon isotope composition in a continuous flow of helium for potassium/argon geochronology is described. Extraction of argon from geological samples in multiple-sample holders was carried out in a chamber by heating with a continuous Nd-YAG laser. The extracted and pre-concentrated argon is passed through a chromatographic capillary column in a flow of helium. Argon is separated from possible contaminants in the column and is injected through an open split into the ion source of an isotope ratio mass spectrometer. Measurement of the (36)Ar, (38)Ar and (40)Ar isotopes was carried out in dynamic mode, using a triple-collector ion detector. These experiments have shown that continuous flow mass spectrometry can be used for the analysis of radiogenic argon in picogram quantities with an accuracy that is satisfactory for the solution of many geochronological problems. The method of argon isotope measurement in a continuous flow of helium is an alternative to the measurement of argon isotopes in the static mode. The sensitivity and accuracy of argon measurement by this method are comparable with those provided by the classical static method. The measurement of argon isotopes in a continuous flow of helium is simpler and more reliable than measurement in the static mode.

Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

BackgroundFor more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation.ResultsIn this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. The results demonstrate significant differences between these four methods.ConclusionIn our hands, the WT-Ovation pico system proposed by Nugen appears to be the most suitable for RNA amplification. This comparative study will be useful to scientists needing to choose an amplification method to carry out microarray experiments involving samples comprising only a few cells and generating picogram amounts of RNA.

Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching

A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition of the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The protein concentration is determined by observing the magnitude of fluorescence altered by quenching the fluorescence on the gold particles in a homogeneous assay format. Under optimal low pH conditions, the assay allowed the determination of picogram quantities (7.0 microg/L) of proteins with an average variation of 4.5% in a 10 min assay. The assay sensitivity was more than 10-fold improved from those of the commonly used most sensitive commercial methods. In addition, the particle sensor provides a simple and rapid assay format without requirements for hazardous test compounds and elevated temperature. Eleven different proteins were tested with the constructed sensor exhibiting a protein-to-protein variability less than 15% allowing protein concentration measurements without the need for recalibration of different proteins.

Digital PCR provides sensitive and absolute calibration for high throughput sequencing

BackgroundNext-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.ResultsWe 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.ConclusionThe 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.

Trail pheromones of ants

Abstract The study of trail laying, recruitment of workers and trail‐following by worker ants comprises a co‐operative study of entomologists and chemists that has resulted in the identification of the chemical nature of such pheromones in many species of five subfamilies of ants. These pheromones may comprise a single compound or, in one exceptional case, a blend of as many as 14 compounds, they may come from a single gland, or in some cases, a combination of two glands. They may be peculiar to a single species or may be shared by a number of species. They exist in the glandular secretion in nanogram to picogram quantities and are detected by workers in minute amounts on a trail. The present state of knowledge of these pheromones and their chemical structures is reviewed. Suitable bioassays and odour perception are discussed and the stereobiology of a few examples is considered.

AcrB et al.: Obstinate contaminants in a picogram scale. One more bottleneck in the membrane protein structure pipeline

Heterologous expression of integral membrane proteins from Helicobacter pylori 26695 in Escherichia coli enabled the identification of 17 candidates for purification and subsequent crystallization. 45% of the purified proteins were contaminated with what was later identified as the multidrug efflux pump (AcrB)of E. coli, and 17% with the succinate dehydrogenase. While additional purification steps ensured removal of succinate dehydrogenase, they failed to remove AcrB completely, leaving picogram amounts present infractions intended for 3D-crystallization. Two of these targets, the Na+ dependent D-glucose/D-galactose transporter (GluP-HP1174) and the carbon starvation protein A (CstA-HP1168), produced small crystals(<40 lm). Crystals from the GluP preparation diffracted to 4.2 A resolution and belonged to the rhombohedral space group H32. Subsequent molecular replacement proved that these crystals were derived from a contaminant, the efflux transporter AcrB. This unexpected crystallization of AcrB from picogram amounts was observed in six new conditions. The systematic occurrence of AcrB in membrane preparations stems from the upregulation of its transcription in response to the stress induced by the expression of a selected target. This, along with its tendency to crystallize in the picogram scale, poses a serious concern in membrane protein expression using heterologous hosts harbouring AcrB.

In-situ detection of single particles of explosive on clothing with confocal Raman microscopy

Confocal Raman microscopy is shown to detect picogram quantities of explosives in-situ on undyed natural and synthetic fibres, and coloured textile specimens leaving potentially evidential materials unaltered. Raman spectra were obtained from pentaerythritol tetranitrate (PETN), trinitrotoluene (TNT), and ammonium nitrate particles trapped between the fibres of the specimens. Despite the presence of spectral bands arising from the natural and synthetic polymers and dyed textiles, the explosive substances could be identified by their characteristic Raman bands. Furthermore, Raman spectra were obtained from explosives particles trapped between highly fluorescent clothing fibres. Raman spectra were collected from explosives particles with maximum dimensions in the range 5–10 μm. Spectra of the explosives on dyed and undyed clothing substrates were readily obtained in-situ within 90 s and without sample preparation.

Behaviour of the Alkaline Earth Elements (Beryllium to Radium) During the Precipitation of Jarosite-Type Compounds

The behaviour of the alkaline earth elements (beryllium, magnesium, calcium, strontium, barium and radium) during the precipitation of jarosite-type compounds was investigated over the range of conditions likely to be encountered in hydrometallurgical processes. Negligible amounts (< 0.05 wt%) of beryllium were incorporated in the structure of either potassium jarosite or sodium jarosite; this was a consequence of the small size of the divalent beryllium ion. In contrast, limited magnesium incorporation seemed to occur in the jarosite structure. Up to 0.20 wt% Mg was incorporated in sodium jarosite and up to 0.38 wt% Mg was detected in potassium jarosite; in both species, the amount increased as the magnesium concentration of the synthesis solution increased. Increasing temperatures, ferric ion concentrations and solution pH had only a minor effect on the extent of magnesium incorporation in the jarosite precipitates. In contrast, increasing concentrations of CuSO4 or ZnSO4 significantly reduced the level of Mg incorporation. Regardless of the calcium concentration of the solution, the calcium contents of the well-washed hydronium jarosite, sodium jarosite, potassium jarosite and lead jarosite precipitates were consistently less than 0.05 wt%Ca. The addition of soluble salts of strontiumand bariumto sulphate processing solutions resulted in the extensive precipitation of SrSO4 and BaSO4. However, electron microprobe analyses detected trace amounts of these elements in the potassium jarosite precipitates, suggesting a modest degree of Sr and Ba solid solubility. Picogram quantities of radium are soluble in sulphate media and 60-90% of the dissolved Ra precipitated with the jarosite-type compounds. However, it is not clear whether the radium was incorporated in the structure of the jarosite or whether it was adsorbed on the jarosite particles.

Microdissection molecular copy‐number counting (µMCC)—unlocking cancer archives with digital PCR

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Carboxylic acid characterization in nanoparticles by thermal desorption chemical ionization mass spectrometry

Recent improvements in the operation of the thermal desorption chemical ionization mass spectrometer allow for the characterization of organic species in 8–40 nm diameter particles. Here we describe the application of this technique to monocarboxylic and dicarboxylic acids, focusing on the response of the instrument to picogram amounts of pure and multicomponent mixtures. Monocarboxylic acids underwent minimal decomposition during analysis, and were identified by the deprotonated parent ion with a sensitivity of 3–8 Hz of integrated ion signal per picogram of sample. Measurements of a binary mixture of monocarboxylic acids showed that desorption and subsequent ionization of these compounds occur independently and have mass-normalized responses identical to pure samples. Dicarboxylic acids appear as the deprotonated parent ion as well as an important decomposition product corresponding to the loss of formic acid from the deprotonated parent. Sensitivities towards these compounds were up to 100 times higher than for the monocarboxylic acids. Experiments using 10–30 nm diameter butanedioic acid particles showed a linear response to collected particulate mass with sufficient sensitivity to support the application of this technique to the characterization of carboxylic acids in ambient atmospheric nanoparticles.

Antibody microarray longevity study: How long‐term storage affects the dynamic range of printed slides

Following development of an antibody microarray we undertook a six month study of a single batch of chips to determine the loss of sensitivity over time. Picogram amounts of capture antibodies are spotted onto epoxy‐silane slides using a Piezorray printer. A single printing, of eleven slides containing 30 anti‐rat antibodies was stored at 4°C in a desiccator until used. A freshly printed slide was analyzed to assure accuracy of print. During the six month test‐period slides were arrayed by first hybridizing with protein standards in separate wells of a pro‐plate system and bound proteins detected using biotin‐conjugated secondary antibodies. Streptavidin conjugated Alexafluor is used to generate the fluorescent signal. A single slide was analyzed with protein standards every two weeks, starting 3 weeks post‐printing and ending at the 6 month time‐point. A single slide was stored at room temperature in a desiccator and tested 3 months post‐printing. We found less than a 2% change in the detection threshold relative to the dynamic range of the standard curves for the slides stored at 4°C. While under the same conditions the slide stored at room temperature exhibited a 15% degradation at 3 months. These findings demonstrate that antibody microarrays can be used for extended periods of time post‐printing, allowing for a single batch of chips to be used for large ongoing studies. Work supported by a grant from Pfizer of Ann Arbor.

Marine methylotrophs revealed by stable‐isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA (<or= 40 kb) and cloned to produce a fosmid library of > 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.

Multistrip Western blotting to increase quantitative data output

The qualitative and quantitative measurements of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multistrip Western blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible, and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.

Highly sensitive, specific determination of 17α-methyl-5β-androstane-3α,17β-diol by gas chromatography coupled to triple mass spectrometry

It is shown that gas chromatography coupled to triple mass spectrometry provides a means of detecting picogram amounts of a methyltestosterone metabolite in biological liquids. The results are compared with results obtained by gas chromatography coupled to high-resolution mass spectrometry

Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas

Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multimutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these 2 anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. Expression GM-CSF in vitro did not alter the infectivity, cytotoxicity, or replication of NV1034 compared to the noncytokine-secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l-6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice, respectively. In these immune-competent models, NV1034 and NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, there was no difference in the antitumor efficacy of these viruses in mice depleted of CD4+ and CD8+ T lymphocytes. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.