Low-yield Samples Research Articles

Exploring Metabolic Characteristics in Different Geographical Locations and Yields of Nicotiana tabacum L. Using Gas Chromatography-Mass Spectrometry Pseudotargeted Metabolomics Combined with Chemometrics.

The quality of crops is closely associated with their geographical location and yield, which is reflected in the composition of their metabolites. Hence, we employed GC-MS pseudotargeted metabolomics to investigate the metabolic characteristics of high-, medium-, and low-yield Nicotiana tabacum (tobacco) leaves from the Bozhou (sweet honey flavour) and Shuicheng (light flavour) regions of Guizhou Province. A total of 124 metabolites were identified and classified into 22 chemical categories. Principal component analysis revealed that the geographical location exerted a greater influence on the metabolic profiling than the yield. Light-flavoured tobacco exhibited increased levels of sugar metabolism- and glycolysis-related intermediate products (trehalose, glucose-6-phosphate, and fructose-6-phosphate) and a few amino acids (proline and leucine), while sweet honey-flavoured tobacco exhibited increases in the tricarboxylic acid cycle (TCA cycle) and the phenylpropane metabolic pathway (p-hydroxybenzoic acid, caffeic acid, and maleic acid). Additionally, metabolite pathway enrichment analysis conducted at different yields and showed that both Shuicheng and Bozhou exhibited changes in six pathways and four of them were the same, mainly C/N metabolism. Metabolic pathway analysis revealed higher levels of intermediates related to glycolysis and sugar, amino acid, and alkaloid metabolism in the high-yield samples, while higher levels of phenylpropane in the low-yield samples. This study demonstrated that GC-MS pseudotargeted metabolomics-based metabolic profiling can be used to effectively discriminate tobacco leaves from different geographical locations and yields, thus facilitating a better understanding of the relationship between metabolites, yield, and geographical location. Consequently, metabolic profiles can serve as valuable indicators for characterizing tobacco yield and geographical location.

Single-Electron Transport and Detection of Graphene Quantum Dots

The integrated structure of graphene single-electron transistor and nanostrip electrometer was prepared using the semiconductor fabrication process. Through the electrical performance test of the large sample number, qualified devices were selected from low-yield samples, which exhibited an obvious Coulomb blockade effect. The results show that the device can deplete the electrons in the quantum dot structure at low temperatures, thus, accurately controlling the number of electrons captured by the quantum dot. At the same time, the nanostrip electrometer coupled with the quantum dot can be used to detect the quantum dot signal, that is, the change in the number of electrons in the quantum dot, because of its quantized conductivity characteristics.

Abstract 2174: A solution for simultaneous DNA & RNA extraction from single tissue without lysate splitting

Abstract Extraction of intact and high quality gDNA or RNA is one of the most common procedures in molecular biology labs. With the advance of NGS, scientists can analyze both genomic and transcriptomic information from the same sample. Techniques that offer the ability to isolate both DNA and RNA from a single biological sample are crucial for this application. There is still great need for improved protocols in this area especially for precious and low-yield samples. Currently, most researchers use TRIzol® to isolate RNA and DNA from the same tube by extracting DNA from the organic phase and RNA from the aqueous phase. Although this method can extract high-quality RNA, the DNA yield is often low. With many biological samples in short supply, it is crucial to maximize yields. We report here using Beckman Coulter reagents, a protocol to isolate both DNA and RNA from the same fresh/frozen sample. The method utilizes size selection to separate DNA and RNA. We tested this method on 4 different frozen mouse tissues and report consistent extraction performance across all 4 tissue types. There are two advantages for this method: (1) There is no need to split a lysate for separate preparation of DNA/RNA; thus, researchers will not lose any portion of DNA or RNA. (2) Due to the different size distributions between DNA and RNA, this method offers quick and efficient extraction. Citation Format: Han Wei. A solution for simultaneous DNA & RNA extraction from single tissue without lysate splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2174.

Terrestrial paleoenvironment characterization across the Permian–Triassic boundary in South China

Well-preserved marine fossils in carbonate rocks permit detailed studies of the end-Permian extinction event in the marine realm. However, the rarity of fossils in terrestrial depositional environments makes it more challenging to attain a satisfactory degree of resolution to describe the biotic turnover on land. Here we present new sedimentological, paleontological and geochemical (X-ray fluorescence) analysis from the study of four terrestrial sections (Chahe, Zhejue, Mide and Jiucaichong) in Western Guizhou and Eastern Yunnan (Yangtze Platform, South China) to evaluate paleoenvironmental changes through the Permian–Triassic transition.Our results show major differences in the depositional environments between the Permian Xuanwei and the Triassic Kayitou formations with a change from fluvial–lacustrine to coastal marine settings. This change is associated with a drastic modification of the preservation mode of the fossil plants, from large compressions to small comminuted debris. Plant fossils spanning the Permian–Triassic boundary show the existence of two distinct assemblages: In the Xuanwei Formation, a Late Permian (Changhsingian) assemblage with characteristic Cathaysian wetland plants (mainly Gigantopteris dictyophylloides, Gigantonoclea guizhouensis, G. nicotianaefolia, G. plumosa, G. hallei, Lobatannularia heinanensis, L. cathaysiana, L. multifolia, Annularia pingloensis, A. shirakii, Paracalamites stenocostatus, Cordaites sp.) is identified. In the lowermost Kayitou Formation, an Early Triassic (Induan) Annalepis–Peltaspermum assemblage is shown, associated with very rare, relictual gigantopterids. Palynological samples are poor, and low yield samples show assemblages almost exclusively represented by spores. A ∼1m thick zone enriched in putative fungal spores was identified near the top of the Xuanwei Formation, including diverse multicellular forms, such as Reduviasporonites sp. This interval likely corresponds to the PTB “fungal spike” conventionally associated with land denudation and ecosystem collapse. While the floral turnover is evident, further studies based on plant diversity would be required in order to assess contribution linked to the end-Permian mass extinction versus local paleoenvironmental changes associated with the transition between the Xuanwei and Kayitou formations.

Optimized whole-genome amplification strategy for extremely AT-biased template.

Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.

Evaluation of High Application Rates of Chemical Conditioners

A very high application rate (935 L/ha) of an aqueous solution of potassium carbonate (15 g/L) plus sodium carbonate (15 g/L) was applied to alfalfa on days having an expectation of good drying weather based on the local forecast. With this application rate, baling moisture (20% w.b.) was achieved on the day of mowing on 78% of the days in low yield (3350 kg dry matter/ha) swaths and 33% of the days in high-yield (5590 kg dry matter/ha) swaths. Drying constants for an exponential drying function averaged 0.427 h1 for treated low-yield samples, 0.314 h1 for treated high-yield samples, 0.272 h1 for nontreated low-yield samples, and 0.217 h1 for nontreated high-yield samples. Flipping of the swath had more effect when combined with chemical conditioning and was more effective with heavy yields than with light yields. Drying data were comparable to values predicted with an established model of alfalfa drying.

505 SEPARATION OF FETAL AND ADULT ERYTHROCYTES BY SELECTIVE HEMOLYSIS: AN AID TO PERNTAL DIAGNOSIS OF HEMOGLOBINOPATHIES

Prenatal diagnosis of hemoglobinopathies is difficult if placental blood samples are contaminated by maternal cells, because β/γ ratios exceed the normal fetal range of 0.07 to 0.15. We have shown that fetal reticulocytes have less carbonic anhydrase than adult cells, and resist NH4Cl-, NH4HCO3-mediated (ørskov) hemolysis. To test this approach in actual cases, pure fetal blood was obtained by fetoscopy from two midtrimester fetuses at risk for homozygous β-thalassemia (thal). 2.5% fetal blood was mixed with blood from the β-thal trait mothers. Samples were incubated with 3H-leucine; an aliquot was treated in the ørskov reaction. Red cell size distributions were analysed with a Coulter channelyzer. Globin chains were separated on carboxymethylcellulose columns. In the mixed samples, only small maternal cells were seen with the channelyzer. Following ørskov hemolysis, only large fetal cells were detected. In case 1, β/γ in the mixed sample was 0.42. β/γ fell to zero following ørskov hemolysis, and was zero in pure fetal blood. This fetus had thal major. In case 2, β/γ was 1.97 before and 0.04 after ørskov hemolysis; it was 0.04 in pure fetal blood. This fetus has β-thal trait. ørskov hemolysis provides pure fetal blood from samples with only 2.5% fetal cells and enables the appropriate diagnoses to be made even when low yield samples are obtained.