Column-based Method Research Articles

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Effective and economical column-based method for RNA isolation from animal cells.

To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass.

A major challenge for microbiome studies is maintaining an even and accurate DNA extraction in the presence of samples with a wide range of bacterial content. Here we compare five DNA extraction methods using replicate stool samples that were diluted to create high and low biomass samples. Our results indicate greater variation in microbiome composition between high and low biomass samples than variation between methods. Many of the extraction methods had reduced yield from low biomass samples; however, our adapted plate column-based extraction method was evenly efficient and captured the largest number of high-quality reads. Based on these results, we have identified a DNA extraction method that ensures adequate yield in metagenomic microbiome studies that have samples with a broad range ofbacterial content.

Diagnosis of Meningococcal Infection Using Internally Controlled Multiplex Real-Time PCR.

Neisseria meningitidis (Nm) is a leading cause of invasive infections associated with high mortality and morbidity, notably meningitis and septicemia. Etiological rapid diagnosis is key for the preventive management of invasive meningococcal disease (IMD). However, conventional methods for diagnosis are time-consuming and could be hampered by the difficulties in culturing the isolates from clinical specimens especially due to early antibiotic treatment. Therefore, sensitive, specific and rapid non-culture-based methods are valuable for early diagnosis, effective therapy, and prevention. Here we describe a real-time PCR multiplex assays for the detection of Nm targeting the meningococcal-specific gene crgA, coding for a LysR-like transcriptional regulator, and six serogroup-specific (A, B, C, W, X, Y) Nm capsular genes, using a Qiagen column-based method for the optimum isolation of DNA from clinical specimens. Internal quality controls were included to monitor extraction of DNA, inhibition and the technical validation of the PCR as well.

CTAB VS. COLUMN-BASED DNA EXTRACTION FROM OLD HERBARIUM MATERIAL

Herbarium specimens have become a major source of information in molecular research of biodiversity. However, getting good DNA samples from old herbarium specimens is still a challenge. The purpose of this project is to test different DNA extraction methods for old material from herbaria that often exhibit high DNA fragmentation. We compared a CTAB-based DNA extraction that is followed by a clean-up with paramagnetic beads with a modified NucleoSpin Plant II protocol, based on silica columns. Our results demonstrate that silica column-based methods have less problems with contamination by polysaccharide and polyphenolic compounds. Taking practical considerations into account, the column-based method is better especially when trying to reduce the amount of leaf tissue used since handling with a tiny pellet makes CTAB difficult.

Using ddPCR to assess the DNA yield of FFPE samples

ObjectivesDetection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples. MethodsWe used 10 μm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10–15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content. ResultsDeparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality. ConclusionsSamples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. The new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits. The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

BackgroundmicroRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes.ResultsWe identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided.ConclusionsDifferent methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that “total RNA” extraction methods do not isolate all types of RNA equally.

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection.

During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of furthermethylation analysis.

A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses.

Laser capture microdissection protocol for gene expression analysis in the brain.

Laser capture microdissection (LCM) allows the isolation of specific cell populations from complex tissues that can be then used for gene expression studies. However, there are no reproducible protocols to study RNA in the brain and, particularly, in the substantia nigra. RNA is a very labile biomolecule that is easily degraded during manipulation. LCM studies use low amounts of material and special precautions must be taken to preserve RNA yield and integrity, which are decisive for PCR analysis. The RNA yield and/or integrity can be affected negatively by tissue manipulation, LCM process and RNA extraction. We have optimized these three critical steps using nigral tissue sections, and developed a LCM protocol to obtain high-quality RNA for gene expression analysis. The optimal LCM protocol requires the use of 20µm-thick tissue sections mounted on glass slides and processed for rapid tyrosine hydroxylase immunofluorescence. Additionally, a total microdissected tissue area of 1mm2 and a column-based RNA extraction method were used to obtain a high RNA yield and integrity. In the rat substantia nigra, we demonstrated the expression of RNA for the angiotensin type 1 and type 2 receptors using this optimized LCM protocol. In conclusion, the LCM protocol reported here can be used to study the expression of both scarcely or abundantly expressed genes in the different brain regions of mammals under both physiological and pathological conditions.

Mutrikuko olatu zentralaren azterketa eta potentzia maximoko puntuaren jarraipena

Susperraldian dauden energia iturri berriztagarri berriak berebiziko garrantzia izatera iritsi dira gaur egungo gizartean. Petrolioarekiko menpekotasuna eta berotegi efektua direla medio, energia lortzeko bide berriak miatu beharra nabarmendu da. Ozeanoek eta itsasoek energia kantite handia pilatzen dute bere baitan. Hala ere, gizakia oraindik ez da gai izan energia honetaz egokiro baliatzeko. Ur-zutabe oszilatzailean oinarritutako metodoa da gaur egun itsasotik energia ateratzeko sistema guztien artetik gehien ikertuenetako bat. Alabaina, lorturiko efizientzia baxuek zaildu egiten dute teknologia honen erabilera. Gaur egungo kontrol-metodo berriek, aldiz, efizientzia hau hobetzea ahalbidetzen dute. Metodo hauen artean badago potentzia maximoko puntuaren jarraipena, batez ere energia eolikoan asko erabilia.

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (≥4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

Improved extraction of humic acids from vermicomposted organic waste by a column-based continuous elution method

ABSTRACTA column-based continuous elution (CCE) method was developed for extracting humic acids (HA) from vermicomposted organic waste. Dissolution of HA by passing extractant (0.2 M NaOH) through vermicompost in a column followed by elution under gravity resulted in yields of 47.5% and 74.7% extractable HA in 24 h by International Humic Substance Society (IHSS) method and CCE method, respectively. These results were 63.3% and 97.9% in 48 h, respectively. The extracted HA showed similar FTIR absorption spectra and chemical compositions. Rate of oxidative degradation of HA during extraction by the CCE method was lower as compared with the IHSS method indicating improvement in extraction without altering the quality.

Column-free magnetic enrichment of pDCs yields significant contamination with DNA-containing dead cells that affect the activation status of the isolated cells

Abstract Despite their role in the induction of adaptive immune responses, plasmacytoid dendritic cells (pDC) play also a crucial role in innate immune defense by recognizing pathogen derived molecular patterns. Unmethylated CpG motifs of bacterial DNA bind specifically endosomal and lysosomal TLR9 in pDCs, thereby inducing secretion of type I IFN or proinflammatory cytokines and classical DC maturation, respectively. However, TLR9 also recognizes CpG motifs in vertebrate DNA of damaged cells as danger-associated molecular patterns (DAMPs) that activate pDCs. For in-vitro research pDCs may be isolated by indirect magnetic cell separation using either Miltenyi Biotec’s column-based or a column-free system from other suppliers. Here we describe the influence of the isolation method on the functionality of the purified pDCs. In addition to the significantly better separation performance, our column-based method enriched 10- and 30-fold less DNA-containing dead cells and debris, respectively. Although both methods yielded pDCs with a comparable phenotype instantly after isolation, after 24h of culture without stimulation pDCs isolated with the column-free system appeared pre-activated as demonstrated by elevated expression of CD80, −83, −86 and HLA-DR and by significantly higher basal IFN-α secretion. Moreover, upon stimulation with CpG-B pDCs isolated with the column-free method induced extraordinary high amounts of IFN-α, whereas pDCs isolated with our system exhibited normal responses to CpG-A and CpG-B. These results clearly demonstrate that unspecific enrichment of dead cells and debris may significantly influence the activation status of the isolated pDCs and therefore affect the results of downstream applications.

GPU-Accelerated Parallel Sparse LU Factorization Method for Fast Circuit Analysis

Lower upper (LU) factorization for sparse matrices is the most important computing step for circuit simulation problems. However, parallelizing LU factorization on the graphic processing units (GPUs) turns out to be a difficult problem due to intrinsic data dependence and irregular memory access, which diminish GPU computing power. In this paper, we propose a new sparse LU solver on GPUs for circuit simulation and more general scientific computing. The new method, which is called GPU accelerated LU factorization (GLU) solver (for GPU LU), is based on a hybrid right-looking LU factorization algorithm for sparse matrices. We show that more concurrency can be exploited in the right-looking method than the left-looking method, which is more popular for circuit analysis, on GPU platforms. At the same time, the GLU also preserves the benefit of column-based left-looking LU method, such as symbolic analysis and column-level concurrency. We show that the resulting new parallel GPU LU solver allows the parallelization of all three loops in the LU factorization on GPUs. While in contrast, the existing GPU-based left-looking LU factorization approach can only allow parallelization of two loops. Experimental results show that the proposed GLU solver can deliver $5.71\times $ and $1.46\times $ speedup over the single-threaded and the 16-threaded PARDISO solvers, respectively, $19.56\times $ speedup over the KLU solver, $47.13\times $ over the UMFPACK solver, and $1.47\times $ speedup over a recently proposed GPU-based left-looking LU solver on the set of typical circuit matrices from the University of Florida (UFL) sparse matrix collection. Furthermore, we also compare the proposed GLU solver on a set of general matrices from the UFL, GLU achieves $6.38\times $ and $1.12\times $ speedup over the single-threaded and the 16-threaded PARDISO solvers, respectively, $39.39\times $ speedup over the KLU solver, $24.04\times $ over the UMFPACK solver, and $2.35\times $ speedup over the same GPU-based left-looking LU solver. In addition, comparison on self-generated $RLC$ mesh networks shows a similar trend, which further validates the advantage of the proposed method over the existing sparse LU solvers.

Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

BackgroundUrinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established.MethodsUEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis.ResultsThe presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method.ConclusionOur results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with appropriate UEV collection procedure. We also suggested that a minimum 250 ml of urine should be processed to gather enough RNA for robust quantification, qualification and downstream analysis.